Mutant Δ8 desaturase genes engineered by targeted mutagenesis and their use in making polyunsaturated fatty acids

ABSTRACT

The present invention relates to mutant Δ8 desaturase genes, which have the ability to convert eicosadienoic acid [20:2 ω-6, EDA] to dihomo-γ-linolenic acid [20:3, DGLA] and/or eicosatrienoic acid [20:3 ω-3, ETrA] to eicosatetraenoic acid [20:3 ω-3, ETA]. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding Δ8 desaturase along with methods of making long-chain polyunsaturated fatty acids (PUFAs) using these mutant Δ8 desaturases in plants and oleaginous yeast are disclosed.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to the creation of nucleic acid fragments encodingmutant Δ8 fatty acid desaturase enzymes and the use of these desaturasesin making long-chain polyunsaturated fatty acids (PUFAs).

BACKGROUND OF THE INVENTION

The importance of PUFAs is undisputed. For example, certain PUFAs areimportant biological components of healthy cells and are considered“essential” fatty acids that cannot be synthesized de novo in mammalsand instead must be obtained either in the diet or derived by furtherdesaturation and elongation of linoleic acid (LA;18:2 ω-6) orα-linolenic acid (ALA; 18:3 ω-3). Additionally PUFA's are constituentsof plasma membranes of cells, where they may be found in such forms asphospholipids or triacylglycerols. PUFA's are necessary for properdevelopment (particularly in the developing infant brain) and for tissueformation and repair and, are precursors to several biologically activeeicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids,leukotrienes, prostaglandins). Studies have shown that a high intake oflong-chain ω-3 PUFAs produces cardiovascular protective effects(Dyerberg, J. et al., Amer. J. Clin. Nutr., 28:958-966 (1975); Dyerberg,J. et al., Lancet, 2(8081):117-119 (Jul. 15, 1978); Shimokawa, H., WorldRev. Nutr. Diet, 88:100-108 (2001); von Schacky, C. and Dyerberg, J.,World Rev. Nutr. Diet, 88:90-99 (2001)). The literature reportswide-ranging health benefits conferred by administration of ω-3 and/orω-6 PUFAs against a variety of symptoms and diseases (e.g., asthma,psoriasis, eczema, diabetes, cancer).

A variety of different hosts including plants, algae, fungi and yeastare being investigated as means for commercial PUFA production. Geneticengineering has demonstrated that the natural abilities of some hostscan be substantially altered to produce various long-chain ω-3/ω-6PUFAs. For example, production of arachidonic acid (ARA; 20:4 ω-6),eicosapentaenoic acid (EPA; 20:5 ω-3) and docosahexaenoic acid (DHA;22:6 ω-3) all require expression of either the Δ9 elongase/Δ8 desaturasepathway or the Δ6 desaturase/Δ6 elongase pathway. The Δ9 elongase/Δ8desaturase pathway is present for example in euglenoid species and ischaracterized by the production of eicosadienoic acid [“EDA”; 20:2 ω-6]and/or eicosatrienoic acid [“ETrA”; 20:3 ω-3]. (FIG. 1). The Δ6desaturase/Δ6 elongase pathway is predominantly found in algae, mosses,fungi, nematodes and humans and is characterized by the production ofγ-linoleic acid [“GLA”; 18:3 ω-6] and/or stearidonic acid [“STA”; 18:4ω-3]) (FIG. 1).

For some applications the Δ9 elongase/Δ8 desaturase pathway is favored.However Δ8 desaturase enzymes are not well known in the art leaving theconstruction of a recombinant Δ9 elongase/Δ8 desaturase pathway withlimited options. The few Δ8 desaturase enzymes identified thus far havethe ability to convert both EDA to dihomo-γ-linolenic acid [20:3, DGLA]and ETrA to eicosatetraenoic acid [20:4, ETA] (wherein ARA are EPA aresubsequently synthesized from DGLA and ETA, respectively, followingreaction with a Δ5 desaturase, while DHA synthesis requires subsequentexpression of an additional C_(20/22) elongase and a Δ4 desaturase).

Several Δ8 desaturase enzymes are known and have been partiallycharacterized (see for example Δ8 desaturases from Euglena gracilisWallis et al., Arch. Biochem. and Biophys., 365(2):307-316 (May 1999);WO 2000/34439; U.S. Pat. No. 6,825,017; WO 2004/057001; WO 2006/012325;WO 2006/012326). Additionally WO 2005/103253 (published Apr. 22, 2005)discloses amino acid and nucleic acid sequences for a Δ8 desaturaseenzyme from Pavlova salina (see also U.S. Publication No. 2005/0273885).Sayanova et al. (FEBS Lett., 580:1946-1952 (2006)) describes theisolation and characterization of a cDNA from the free living soilamoeba Acanthamoeba castellanii that, when expressed in Arabidopsis,encodes a C₂₀ Δ8 desaturase. Furthermore, commonly owned and U.S.Provisional Application No. 60/795,810 filed Apr. 28, 2006 disclosesamino acid and nucleic acid sequences for a Δ8 desaturase enzyme fromPavlova lutheri (CCMP459).

A need remains therefore for additional Δ8 desaturase enzymes to be usedin recombinant pathways for the production of PUFA's. Applicants havesolved the stated need by developing a synthetically engineered mutantEuglena gracilis Δ8 desaturase.

SUMMARY OF THE INVENTION

The present invention relates to new recombinant constructs encodingmutant polypeptides having Δ8 desaturase activity, and their use inplants and yeast for the production of PUFAs and particularly ω-3 and/orω-6 fatty acids.

Accordingly the invention provides, an isolated polynucleotidecomprising: (a) a nucleotide sequence encoding a mutant polypeptidehaving Δ8 desaturase activity having an amino acid sequence as set forthin SEQ ID NO:2 and wherein SEQ ID NO:2 is not identical to SEQ ID NO:10;or, (b) a complement of the nucleotide sequence of part (a), wherein thecomplement and the nucleotide sequence consist of the same number ofnucleotides and are 100% complementary.

In an alternate embodiment the invention provides an isolatedpolynucleotide comprising: (a) a nucleotide sequence encoding a mutantpolypeptide having Δ8 desaturase activity, having an amino acid sequenceas set forth in SEQ ID NO:198 and wherein SEQ ID NO:198 is not identicalto SEQ ID NO:10; or, (b) a complement of the nucleotide sequence of part(a), wherein the complement and the nucleotide sequence consist of thesame number of nucleotides and are 100% complementary.

It is one aspect of the invention to provide polypeptides encoded by thepolynucleotides of the invention as well as genetic chimera and hostcells transformed and expressing the same.

In another aspect the invention provides a method for making long-chainpolyunsaturated fatty acids in a yeast cell comprising: (a) providing ayeast cell of the invention; and (b) growing the yeast cell of (a) underconditions wherein long-chain polyunsaturated fatty acids are produced.

In another aspect of the invention provides microbial oil obtained fromthe yeast of the invention.

In an alternate embodiment the invention provides an oleaginous yeastproducing at least about 25% of its dry cell weight as oil comprising:

a) a first recombinant DNA construct comprising an isolatedpolynucleotide encoding a Δ8 desaturase polypeptide of the inventionoperably linked to at least one regulatory sequence; and,

b) at least one second recombinant DNA construct comprising an isolatedpolynucleotide operably linked to at least one regulatory sequence, theconstruct encoding a polypeptide selected from the group consisting of:a Δ4 desaturase, a Δ5 desaturase, Δ6 desaturase, a Δ9 desaturase, a Δ12desaturase, a Δ15 desaturase, a Δ17 desaturase, a Δ9 elongase, aC_(14/16) elongase, a C_(16/18) elongase, a C_(18/20) elongase and aC_(20/22) elongase.

In another aspect the invention provides a food or feed productcomprising the microbial oil of the invention.

In another embodiment the invention provides a method for producingdihomo-γ-linoleic acid comprising:

-   -   a) providing an oleaginous yeast comprising:        -   (i) a recombinant construct encoding a Δ8 desaturase            polypeptide having an amino acid sequence as set forth in            SEQ ID NO:2, wherein SEQ ID NO:2 is not identical to SEQ ID            NO:10; and,        -   (ii) a source of eicosadienoic acid;    -   b) growing the yeast of step (a) under conditions wherein the        recombinant construct encoding a Δ8 desaturase polypeptide is        expressed and eicosadienoic acid is converted to        dihomo-γ-linoleic acid, and;    -   c) optionally recovering the dihomo-γ-linoleic acid of step (b).

In an alternate embodiment the invention provides a method for producingeicosatetraenoic acid comprising:

-   -   a) providing an oleaginous yeast comprising:        -   (i) a recombinant construct encoding a Δ8 desaturase            polypeptide having an amino acid sequence as set forth in            SEQ ID NO:2, wherein SEQ ID NO:2 is not identical to SEQ ID            NO:10; and,        -   (ii) a source of eicosatrienoic acid;    -   b) growing the yeast of step (a) under conditions wherein the        recombinant construct encoding a Δ8 desaturase polypeptide is        expressed and eicosatrienoic acid is converted to        eicosatetraenoic acid, and;    -   c) optionally recovering the eicosatetraenoic acid of step (b).

In another embodiment the invention provides a method for the productionof dihomo-γ-linoleic acid comprising:

-   -   a) providing a yeast cell comprising:        -   i) a first recombinant DNA construct comprising the isolated            polynucleotide of the invention operably linked to at least            one regulatory sequence, and;        -   ii) at least one second recombinant DNA construct comprising            an isolated polynucleotide encoding a Δ9 elongase            polypeptide, operably linked to at least one regulatory            sequence;    -   b) providing the yeast cell of (a) with a source of linolenic        acid, and;    -   c) growing the yeast cell of (b) under conditions where        dihomo-γ-linoleic acid is formed.

Biological Deposits

The following plasmid has been deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209,and bears the following designation, Accession Number and date ofdeposit (Table 1).

TABLE 1 ATCC Deposits Plasmid Accession Number Date of Deposit pKR72PTA-6019 May 28, 2004

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

FIG. 1 is a representative PUFA biosynthetic pathway.

FIG. 2 shows a topological model of EgD8S.

FIG. 3 shows an alignment of EgD8S (SEQ ID NO:10), a Δ6 desaturase ofAmylomyces rouxii (SEQ ID NO:13), a Δ6 desaturase of Rhizopus orizae(SEQ ID NO:14), a Δ8 fatty acid desaturase-like protein of Leishmaniamajor (GenBank Accession No. CAJ09677; SEQ ID NO:15), and a Δ6desaturase of Mortierella isabellina (GenBank Accession No. AAG38104;SEQ ID NO:16). The method of alignment used corresponds to the “ClustalW method of alignment”.

FIG. 4 shows an alignment of EgD8S (SEQ ID NO:10), the cytochrome b₅ ofSaccharomyces cerevisiae (GenBank Accession No. P40312; SEQ ID NO:178)and a probable cytochrome b₅ 1 of Schizosaccharomyces pombe (GenBankAccession No. 094391; SEQ ID NO:179). The method of alignment usedcorresponds to the “Clustal W method of alignment”.

FIG. 5 provides plasmid maps for the following: (A) pZKLeuN-29E3; and,(B) pY116.

FIG. 6 provides plasmid maps for the following: (A) pKUNFmkF2; (B)pDMW287F; (C) pDMW214; and, (D) pFmD8S.

FIG. 7 diagrams the synthesis of the Mutant EgD8S-5B, by ligation offragments from Mutant EgD8S-1 and Mutant EgD8S-2B.

FIG. 8A diagrams the synthesis of Mutant EgD8S-008, by ligation offragments from Mutant EgD8S-001 and Mutant EgD8S-003. Similarly, FIG. 8Bdiagrams the synthesis of Mutant EgD8S-009, by ligation of fragmentsfrom Mutant EgD8S-001 and Mutant EgD8S-004.

FIG. 9A diagrams the synthesis of Mutant EgD8S-013, by ligation offragments from Mutant EgD8S-009 and Mutant EgD8S-23. Similarly, FIG. 9Bdiagrams the synthesis of Mutant EgD8S-015, by ligation of fragmentsfrom Mutant EgD8S-008 and Mutant EgD8S-28.

FIG. 10 shows an alignment of EgD8S (SEQ ID NO:10), Mutant EgD8S-23 (SEQID NO:4), Mutant EgD8S-013 (SEQ ID NO:6) and Mutant EgD8S-015 (SEQ IDNO:8). The method of alignment used corresponds to the “Clustal W methodof alignment”.

FIG. 11 provides plasmid maps for the following: (A) pKo2UFm8; and, (B)pKO2UF8289.

FIG. 12 provides a plasmid map for pKR1060.

FIG. 13 provides a plasmid map for pKR1059.

FIG. 14 shows the lipid profiles of somatic soybean embryos expressingEgD8S-23 and the Euglena gracilis delta-9 elongase for the top 5 events(see example 17).

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

A Sequence Listing is provided herewith on Compact Disk. The contents ofthe Compact Disk containing the Sequence Listing are hereby incorporatedby reference in compliance with 37 CFR 1.52(e). The Compact Disks aresubmitted in triplicate and are identical to one another. The disks arelabeled “Copy 1—Sequence Listing”, “Copy 2—Sequence Listing”, and CRF.The disks contain the following file: CL3495 SeqListing_(—)11.27.06_ST25 having the following size: 293,000 bytes andwhich was created Dec. 6, 2006.

SEQ ID NOs:1-17, 19-23, 165 and 172-177 are ORFs encoding genes orproteins (or portions thereof) or plasmids, as identified in Table 2.

TABLE 2 Summary Of Nucleic Acid And Protein SEQ ID Numbers Nucleic acidDescription and Abbreviation SEQ ID NO. Protein SEQ ID NO. Syntheticmutant Δ8 desaturase, derived from  1  2 Euglena gracilis(“EgD8S-consensus”) optionally (1272 bp) (422 AA) comprising M1, M2, M3,M8, M12, M15, M16, M18, M21, M26, M38, M45, M46, M51, M63, M68, M69 andM70 mutation sites Synthetic mutant Δ8 desaturase, derived from 197 198Euglena gracilis (“EgD8S-consensus”) optionally (1272 bp) (422 AA)comprising M1, M2, M3, M6, M8, M12, M14, M15, M16, M18, M19, M21, M22,M26, M38, M39, M40, M41, M45, M46, M49, M50, M51, M53, M54, M58, M63,M68, M69 and M70 mutation sites Synthetic mutant Δ8 desaturase, derived 3  4 from Euglena gracilis (“Mutant EgD8S-23”) (1272 bp) (422 AA)Synthetic mutant Δ8 desaturase, derived  5  6 from Euglena gracilis(“Mutant EgD8S- (1272 bp) (422 AA) 013”) Synthetic mutant Δ8 desaturase,derived  7  8 from Euglena gracilis (“Mutant EgD8S- (1272 bp) (422 AA)015”) Synthetic Δ8 desaturase, derived from  9  10 Euglena gracilis,codon-optimized for (1272 bp) (422 AA) expression in Yarrowia lipolytica(“EgD8S”) Euglena gracilis Δ8 desaturase (full-length  11  12 gene isnucleotides 4-1269 (Stop)) (1271 bp) (421 AA) (“EgD8”) Amylomyces rouxiiΔ6 desaturase —  13 (GenBank Accession No. AAR27297) (467 AA) Rhizopusorizae Δ6 desaturase (GenBank —  14 Accession No. AAS93682) (445 AA)Leishmania major Δ8 fatty acid —  15 desaturase-like protein (GenBank(382 AA) Accession No. CAJ09677) Mortierella isabellina Δ6 desaturase — 16 (GenBank Accession No. AAG38104) (439 AA) Saccharomyces cerevisiaecytochrome b₅ — 178 (GenBank Accession No. P40312) (120 AA)Schizosaccharomyces pombe probable — 179 cytochrome b₅ 1 (GenBankAccession No. (124 AA) O94391) Plasmid pZKLeuN-29E3  17 — (14,655 bp)  Synthetic C_(16/18) elongase gene derived  19 — from Mortierella alpinaELO3, codon-  (828 bp) optimized for expression in Yarrowia lipolyticaPlasmid pFmD8S  20 — (8,910 bp)  Plasmid pKUNFmkF2  21 — (7,145 bp) Plasmid pDMW287F  22 — (5,473 bp)  Plasmid pDMW214  23 — (9,513 bp) Plasmid pKO2UFkF2 165 — (8,560 bp)  Isochrysis galbana Δ9 elongase(GenBank 172 173 Accession No. AF390174) (1064 bp) (263 AA) Synthetic Δ9elongase gene derived from 174 173 Isochrysis galbana, codon-optimizedfor  (792 bp) (263 AA) expression in Yarrowia lipolytica Euglenagracills Δ9 elongase 175 176  (777 bp) (258 AA) Synthetic Δ9 elongasegene derived from 177 176 Euglena gracilis, codon-optimized for  (777bp) (258 AA) expression in Yarrowia lipolytica Plasmid pY116 180 — (8739bp) Plasmid pKO2UF8289 181 — (15,304 bp)   Synthetic mutant Δ8desaturase, derived 182 — from Euglena gracilis (“modified Mutant (1288bp) EgD8S-23”), comprising a 5′ Not1 site Plasmid pKR457 183 — (5252 bp)Plasmid pKR1058 184 — (6532 bp) Plasmid pKR607 185 — (7887 bp) PlasmidpKR1060 186 — (11,766 bp)   Plasmid pKR906 189 — (4311 bp) Plasmid pKR72190 — (7085 bp) Plasmid pKR1010 191 — (7873 bp) Plasmid pKR1059 192 —(11752 bp)  Euglena gracilis Δ9 elongase - 5′ 194 — sequence of the cDNAinsert from clone  (757 bp) eeg1c.pk001.n5.f. Euglena gracilis Δ9elongase - 3′ 195 — sequence of the cDNA insert from clone  (774 bp)eeg1c.pk001.n5.f. Euglena gracilis Δ9 elongase - sequence 196 — alignedfrom SEQ ID NO: 1 and SEQ ID (1201 bp) NO: 2 (full cDNA sequenceexcluding polyA tail)

SEQ ID NO:18 corresponds to a LoxP recombination site that is recognizedby a Cre recombinase enzyme.

SEQ ID NOs:24-164 correspond to 70 pairs of nucleotide primers (i.e.,1A, 1B, 2A, 2B, 3A, 3B, etc. up to 69A, 69B, 70A and 70B, respectively),used to create specific targeted mutations at mutation sites M1, M2, M3,etc. up to M70.

SEQ ID NOs:166-171 correspond to His-rich motifs that are featured inmembrane-bound fatty acid desaturases belonging to a super-family ofmembrane di-iron proteins.

SEQ ID NOs:187 and 188 correspond to primers oEugEL1-1 and oEugEL1-2,respectively, used to amplify a Euglena gracilis Δ9 elongase.

SEQ ID NO:193 is the M13F universal primer.

DETAILED DESCRIPTION OF THE INVENTION

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

All patents, patent applications, and publications cited herein areincorporated by reference in their entirety. This specifically includesthe following commonly owned and applications: U.S. patent applicationSer. No. 10/840,478, Ser. No. 10/840,579 and Ser. No. 10/840,325 (filedMay 6, 2004), U.S. patent application Ser. No. 10/869,630 (filed Jun.16, 2004), U.S. patent application Ser. No. 10/882,760 (filed Jul. 1,2004), U.S. patent application Ser. No. 10/985,254 and Ser. No.10/985,691 (filed Nov. 10, 2004), U.S. patent application Ser. No.10/987,548 (filed Nov. 12, 2004), U.S. patent application Ser. No.11/024,545 and Ser. No. 11/024,544 (filed Dec. 29, 2004), U.S. patentapplication Ser. No. 11/166,993 (filed Jun. 24, 2005), U.S. patentapplication Ser. No. 11/183,664 (filed Jul. 18, 2005), U.S. patentapplication Ser. No. 11/185,301 (filed Jul. 20, 2005), U.S. patentapplication Ser. No. 11/190,750 (filed Jul. 27, 2005), U.S. patentapplication Ser. No. 11/198,975 (filed Aug. 8, 2005), U.S. patentapplication Ser. No. 11/225,354 (filed Sep. 13, 2005), U.S. patentapplication Ser. No. 11/251,466 (filed Oct. 14, 2005), U.S. patentapplication Ser. No. 11/254,173 and Ser. No. 11/253,882 (filed Oct. 19,2005), U.S. patent application Ser. No. 11/264,784 and Ser. No.11/264,737 (filed Nov. 1, 2005), U.S. patent application Ser. No.11/265,761 (filed Nov. 2, 2005), U.S. Patent Application No. 60/739,989(filed Nov. 23, 2005), U.S. Patent Application No. 60/793,575 (filedApr. 20, 2006), U.S. Patent Application No. 60/795,810 (filed Apr. 28,2006), U.S. Patent Application No. 60/796,637 (filed May 1, 2006) andU.S. Patent Applications No. 60/801,172 and No. 60/801,119 (filed May17, 2006).

Additionally, commonly owned U.S. patent application Ser. No.10/776,311, (published Aug. 26, 2004) relating to the production ofPUFAs in plants, and U.S. patent application Ser. No. 10/776,889(published Aug. 26, 2004) relating to annexin promoters and their use inexpression of transgenes in plants, are incorporated by reference intheir entirety.

The present invention provides mutant Δ8 desaturase enzymes and genesencoding the same, that may be used for the manipulation of biochemicalpathways for the production of healthful PUFAs.

PUFAs, or derivatives thereof, made by the methodology disclosed hereincan be used as dietary substitutes, or supplements, particularly infantformulas, for patients undergoing intravenous feeding or for preventingor treating malnutrition. Alternatively, the purified PUFAs (orderivatives thereof) may be incorporated into cooking oils, fats ormargarines formulated so that in normal use the recipient would receivethe desired amount for dietary supplementation. PUFAs may also be usedas anti-inflammatory or cholesterol lowering agents as components ofpharmaceutical or veterinary compositions.

DEFINITIONS

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

“Triacylglycerols” are abbreviated TAGs.

The term “fatty acids” refers to long-chain aliphatic acids (alkanoicacids) of varying chain lengths, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂. Additional details concerning thedifferentiation between “saturated fatty acids” versus “unsaturatedfatty acids”, “monounsaturated fatty acids” versus “polyunsaturatedfatty acids” (or “PUFAs”), and “omega-6 fatty acids” (ω-6 or n-6) versus“omega-3 fatty acids” (ω-3 or n-3) are provided in WO2004/101757.

Fatty acids are described herein by a simple notation system of “X:Y”,wherein X is the number of carbon (C) atoms in the particular fatty acidand Y is the number of double bonds. The number following the fatty aciddesignation indicates the position of the double bond from the carboxylend of the fatty acid with the “c” affix for the cis-configuration ofthe double bond [e.g., palmitic acid (16:0), stearic acid (18:0), oleicacid (18:1, 9c), petroselinic acid (18:1, 6c), LA (18:2, 9c, 12c), GLA(18:3, 6c, 9c, 12c) and ALA (18:3, 9c, 12c, 15c)]. Unless otherwisespecified, 18:1, 18:2 and 18:3 refer to oleic, LA and ALA fatty acids.If not specifically written as otherwise, double bonds are assumed to beof the cis configuration. For instance, the double bonds in 18:2 (9,12)would be assumed to be in the cis configuration.

Nomenclature used to describe PUFAs in the present disclosure is shownbelow in Table 3. In the column titled “Shorthand Notation”, theomega-reference system is used to indicate the number of carbons, thenumber of double bonds and the position of the double bond closest tothe omega carbon, counting from the omega carbon (which is numbered 1for this purpose). The remainder of the Table summarizes the commonnames of ω-3 and ω-6 fatty acids and their precursors, the abbreviationsthat will be used throughout the specification, and each compounds'chemical name.

TABLE 3 Nomenclature Of Polyunsaturated Fatty Acids And PrecursorsCommon Shorthand Name Abbreviation Chemical Name Notation Myristic —tetradecanoic 14:0 Palmitic Palmitate hexadecanoic 16:0 Palmitoleic —9-hexadecenoic 16:1 Stearic — octadecanoic 18:0 Oleic —cis-9-octadecenoic 18:1 Linoleic LA cis-9,12-octadecadienoic 18:2 ω-6γ-Linoleic GLA cis-6,9,12- 18:3 ω-6 octadecatrienoic Eicosadienoic EDAcis-11,14-eicosadienoic 20:2 ω-6 Dihomo-γ- DGLAcis-8,11,14-eicosatrienoic 20:3 ω-6 Linoleic Arachidonic ARAcis-5,8,11,14- 20:4 ω-6 eicosatetraenoic α-Linolenic ALA cis-9,12,15-18:3 ω-3 octadecatrienoic Stearidonic STA cis-6,9,12,15- 18:4 ω-3octadecatetraenoic Eicosatrienoic ETrA cis-11,14,17- 20:3 ω-3eicosatrienoic Eicosatetraenoic ETA cis-8,11,14,17- 20:4 ω-3eicosatetraenoic Eicosapentaenoic EPA cis-5,8,11,14,17- 20:5 ω-3eicosapentaenoic Docosapentaenoic DPA cis-7,10,13,16,19- 22:5 ω-3docosapentaenoic Docosahexaenoic DHA cis-4,7,10,13,16,19- 22:6 ω-3docosahexaenoic

The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipidscomposed of three fatty acyl residues esterified to a glycerol molecule(and such terms will be used interchangeably throughout the presentdisclosure herein). Such oils can contain long-chain PUFAs, as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. Thus, “oil biosynthesis” generically refers to thesynthesis of TAGs in the cell.

“Percent (%) PUFAs in the total lipid and oil fractions” refers to thepercent of PUFAs relative to the total fatty acids in those fractions.The term “total lipid fraction” or “lipid fraction” both refer to thesum of all lipids (i.e., neutral and polar) within an oleaginousorganism, thus including those lipids that are located in thephosphatidylcholine (PC) fraction, phosphatidyletanolamine (PE) fractionand triacylglycerol (TAG or oil) fraction. However, the terms “lipid”and “oil” will be used interchangeably throughout the specification.

The term “PUFA biosynthetic pathway” refers to a metabolic process thatconverts oleic acid to LA, EDA, GLA, DGLA, ARA, ALA, STA, ETrA, ETA,EPA, DPA and DHA. This process is well described in the literature(e.g., see WO 2006/052870). Briefly, this process involves elongation ofthe carbon chain through the addition of carbon atoms and desaturationof the molecule through the addition of double bonds, via a series ofspecial desaturation and elongation enzymes (i.e., “PUFA biosyntheticpathway enzymes”) present in the endoplasmic reticulim membrane. Morespecifically, “PUFA biosynthetic pathway enzyme” refers to any of thefollowing enzymes (and genes which encode said enzymes) associated withthe biosynthesis of a PUFA, including: a Δ4 desaturase, a Δ5 desaturase,a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, aΔ9 desaturase, a Δ8 desaturase, a C_(14/16) elongase, a Δ9 elongase, aC_(16/18) elongase, a C_(18/20) elongase and/or a C_(20/22) elongase.

The term “ω-3/ω-6 fatty acid biosynthetic pathway” refers to a set ofgenes which, when expressed under the appropriate conditions encodeenzymes that catalyze the production of either or both ω-3 and ω-6 fattyacids. Typically the genes involved in the ω-3/ω-6 fatty acidbiosynthetic pathway encode some or all of the following enzymes: Δ12desaturase, Δ6 desaturase, C_(18/20) elongase, C_(20/22) elongase, Δ5desaturase, Δ17 desaturase, Δ15 desaturase, Δ9 desaturase, Δ8desaturase, a Δ9 elongase and Δ4 desaturase. A representative pathway isillustrated in FIG. 1, providing for the conversion of oleic acidthrough various intermediates to DHA, which demonstrates how both ω-3and ω-6 fatty acids may be produced from a common source. The pathway isnaturally divided into two portions where one portion will generate ω-3fatty acids and the other portion, only ω-6 fatty acids. That portionthat only generates ω-3 fatty acids will be referred to herein as theω-3 fatty acid biosynthetic pathway, whereas that portion that generatesonly ω-6 fatty acids will be referred to herein as the ω-6 fatty acidbiosynthetic pathway.

The term “functional” as used herein in context with the ω-3/ω-6 fattyacid biosynthetic pathway means that some (or all of) the genes in thepathway express active enzymes, resulting in in vivo catalysis orsubstrate conversion. It should be understood that “ω-3/ω-6 fatty acidbiosynthetic pathway” or “functional ω-3/ω-6 fatty acid biosyntheticpathway” does not imply that all the genes listed in the above paragraphare required, as a number of fatty acid products will only require theexpression of a subset of the genes of this pathway.

The term “desaturase” refers to a polypeptide that can desaturate, i.e.,introduce a double bond, in one or more fatty acids to produce a fattyacid or precursor of interest. Despite use of the omega-reference systemthroughout the specification to refer to specific fatty acids, it ismore convenient to indicate the activity of a desaturase by countingfrom the carboxyl end of the substrate using the delta-system. Ofparticular interest herein are Δ8 desaturases that will desaturate afatty acid between the 8^(th) and 9^(th) carbon atom numbered from thecarboxyl-terminal end of the molecule and that can, for example,catalyze the conversion of EDA to DGLA and/or ETrA to ETA. Otherdesaturases include: 1.) Δ5 desaturases that catalyze the conversion ofDGLA to ARA and/or ETA to EPA; 2.) Δ6 desaturases that catalyze theconversion of LA to GLA and/or ALA to STA; 3.) Δ4 desaturases thatcatalyze the conversion of DPA to DHA; 4.) Δ12 desaturases that catalyzethe conversion of oleic acid to LA; 5.) Δ15 desaturases that catalyzethe conversion of LA to ALA and/or GLA to STA; 6.) Δ17 desaturases thatcatalyze the conversion of ARA to EPA and/or DGLA to ETA; and 7.) Δ9desaturases that catalyze the conversion of palmitate to palmitoleicacid (16:1) and/or stearate to oleic acid (18:1). In the art, Δ15 andΔ17 desaturases are also occasionally referred to as “omega-3desaturases”, “w-3 desaturases”, and/or “ω-3 desaturases”, based ontheir ability to convert ω-6 fatty acids into their ω-3 counterparts(e.g., conversion of LA into ALA and ARA into EPA, respectively). Insome embodiments, it is most desirable to empirically determine thespecificity of a particular fatty acid desaturase by transforming asuitable host with the gene for the fatty acid desaturase anddetermining its effect on the fatty acid profile of the host.

For the purposes herein, the term “EgD8” refers to a Δ8 desaturaseenzyme (SEQ ID NO:12) isolated from Euglena gracilis, encoded by SEQ IDNO:11 herein. EgD8 is 100% identical and functionally equivalent to“Eg5”, as described in WO 2006/012325 and WO 2006/012326[US2005-0287652-A1].

Similarly, the term “EgD8S” refers to a synthetic Δ8 desaturase derivedfrom Euglena gracilis that is codon-optimized for expression in Yarrowialipolytica herein (i.e., SEQ ID NOs:9 and 10). EgD8S is 100% identicaland functionally equivalent to “D8SF”, as described in WO 2006/012325and WO 2006/012326.

The term “mutant EgD8S” refers to a Δ8 desaturase of the presentinvention that has at least one mutation with respect to the syntheticΔ8 desaturase derived from Euglena gracilis that is codon-optimized forexpression in Yarrowia lipolytica (i.e., EgD8S). Although “mutations”may include any deletions, insertions and point mutations (orcombinations thereof), in preferred embodiments the mutant EgD8S is setforth in SEQ ID NO:2, wherein: (i) SEQ ID NO:2 comprises at least onemutation selected from the group consisting of: 4S to A, 5K to S, 12T toV, 16T to K, 17T to V, 54A to G, 55F to Y, 66P to Q, 67S to A, 108S toL, 117G to A, 118Y to F, 120L to M, 121M to L, 125Q to H, 126M to L,132V to L, 133L to V, 162L to V, 163V to L, 170G to A, 171L to V, 279Tto L, 280L to T, 293L to M, 3461 to V, 3471 to L, 348T to S, 407A to S,408V to Q, 418A to G, 419G to A and 422L to Q, wherein the mutations aredefined with respect to the synthetic codon-optimized sequence of EgD8S(i.e., SEQ ID NO:10); and (ii) SEQ ID NO:2 is not 100% identical to SEQID NO:10. In more preferred embodiments, the mutant EgD8S has at leastabout 10-18 mutations with respect to the synthetic codon-optimizedsequence of EgD8S, more preferably at least about 19-25 mutations, andmost preferably at least about 26-33 mutations with respect to syntheticcodon-optimized sequence of EgD8S (i.e., SEQ ID NO:10). In anotherembodiment, the Δ8 desaturase activity of the mutant EgD8S is at leastabout functionally equivalent to the Δ8 desaturase activity of thesynthetic codon-optimized EgD8S (SEQ ID NO:10).

A mutant EgD8S is “at least about functionally equivalent” to EgD8S whenenzymatic activity and specific selectivity of the mutant EgD8S sequenceare comparable to that of EgD8S, despite differing polypeptidesequences. Thus, a functionally equivalent mutant EgD8S sequence willpossess Δ8 desaturase activity that is not substantially reduced withrespect to that of EgD8S when the “conversion efficiency” of each enzymeis compared (i.e., a mutant EgD8S will have at least about 50%,preferably at least about 75%, more preferably at least about 85%, andmost preferably at least about 95% of the enzymatic activity of EgD8S).In more preferred embodiments, the mutant EgD8S will have increasedenzymatic activity and specific selectivity when compared to that ofEgD8S (i.e., at least about 105%, more preferably at least about 115%and most preferably at least about 125% of the enzymatic activity ofEgD8S).

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme (e.g., adesaturase) can convert substrate to product. The conversion efficiencyis measured according to the following formula:([product]/[substrate+product])*100, where ‘product’ includes theimmediate product and all products in the pathway derived from it.

The term “elongase system” refers to a suite of four enzymes that areresponsible for elongation of a fatty acid carbon chain to produce afatty acid that is 2 carbons longer than the fatty acid substrate thatthe elongase system acts upon. More specifically, the process ofelongation occurs in association with fatty acid synthase, whereby CoAis the acyl carrier (Lassner et al., The Plant Cell, 8:281-292 (1996)).In the first step, which has been found to be both substrate-specificand also rate-limiting, malonyl-CoA is condensed with a long-chainacyl-CoA to yield CO₂ and a β-ketoacyl-CoA (where the acyl moiety hasbeen elongated by two carbon atoms). Subsequent reactions includereduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA and a secondreduction to yield the elongated acyl-CoA. Examples of reactionscatalyzed by elongase systems are the conversion of GLA to DGLA, STA toETA and EPA to DPA.

For the purposes herein, an enzyme catalyzing the first condensationreaction (i.e., conversion of malonyl-CoA to β-ketoacyl-CoA) will bereferred to generically as an “elongase”. In general, the substrateselectivity of elongases is somewhat broad but segregated by both chainlength and the degree of unsaturation. Accordingly, elongases can havedifferent specificities. For example, a C_(14/16) elongase will utilizea C₁₄ substrate (e.g., myristic acid), a C_(16/18) elongase will utilizea C₁₆ substrate (e.g., palmitate), a C_(18/20) elongase will utilize aC₁₈ substrate (e.g., GLA, STA) and a C_(20/22) elongase will utilize aC₂₀ substrate (e.g., EPA). In like manner, a Δ9 elongase is able tocatalyze the conversion of LA and ALA to EDA and ETrA, respectively(e.g., WO 2002/077213). It is important to note that some elongases havebroad specificity and thus a single enzyme may be capable of catalyzingseveral elongase reactions (e.g., thereby acting as both a C_(16/18)elongase and a C_(18/20) elongase).

The term “Δ9 elongase/Δ8 desaturase pathway” refers to a biosyntheticpathway for production of long-chain PUFAs, said pathway minimallycomprising a Δ9 elongase and a Δ8 desaturase and thereby enablingbiosynthesis of DGLA and/or ETA from LA and ALA, respectively. Thispathway may be advantageous in some embodiments, as the biosynthesis ofGLA and/or STA is excluded.

The term “amino acid” will refer to the basic chemical structural unitof a protein or polypeptide. The amino acids are identified by eitherthe one-letter code or the three-letter codes for amino acids, inconformity with the IUPAC-IYUB standards described in Nucleic AcidsResearch, 13:3021-3030 (1985) and in the Biochemical Journal, 219(2):345-373 (1984), which are herein incorporated by reference.

The term “conservative amino acid substitution” refers to a substitutionof an amino acid residue in a given protein with another amino acid,without altering the chemical or functional nature of that protein. Forexample, it is well known in the art that alterations in a gene thatresult in the production of a chemically equivalent amino acid at agiven site (but do not affect the structural and functional propertiesof the encoded, folded protein) are common. For the purposes of thepresent invention, “conservative amino acid substitutions” are definedas exchanges within one of the following five groups:

-   -   1. Small aliphatic, nonpolar or slightly polar residues: Ala        [A], Ser [S], Thr [T] (Pro [P], Gly [G]);    -   2. Polar, negatively charged residues and their amides: Asp [D],        Asn [N], Glu [E], Gln [Q];    -   3. Polar, positively charged residues: His [H], Arg [R], Lys        [K];    -   4. Large aliphatic, nonpolar residues: Met [M], Leu [L], Ile        [I], Val M (Cys [C]); and    -   5. Large aromatic residues: Phe [F], Tyr [Y], Trp [W].        Thus, Ala, a slightly hydrophobic amino acid, may be substituted        by another less hydrophobic residue (e.g., Gly). Similarly,        changes which result in substitution of one negatively charged        residue for another (e.g., Asp for Glu) or one positively        charged residue for another (e.g., Lys for Arg) can also be        expected to produce a functionally equivalent product. As such,        conservative amino acid substitutions generally maintain: 1.)        the structure of the polypeptide backbone in the area of the        substitution; 2.) the charge or hydrophobicity of the molecule        at the target site; or 3.) the bulk of the side chain.        Additionally, in many cases, alterations of the N-terminal and        C-terminal portions of the protein molecule would also not be        expected to alter the activity of the protein.

The term “non-conservative amino acid substitution” refers to an aminoacid substitution that is generally expected to produce the greatestchange in protein properties. Thus, for example, a non-conservativeamino acid substitution would be one whereby: 1.) a hydrophilic residueis substituted for/by a hydrophobic residue (e.g., Ser or Thr for/byLeu, Ile, Val); 2.) a Cys or Pro is substituted for/by any otherresidue; 3.) a residue having an electropositive side chain issubstituted for/by an electronegative residue (e.g., Lys, Arg or Hisfor/by Asp or Glu); or, 4.) a residue having a bulky side chain issubstituted for/by one not having a side chain (e.g., Phe for/by Gly).Sometimes, non-conservative amino acid substitutions between two of thefive groups will not affect the activity of the encoded protein.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, “nucleic acid fragment” and “isolated nucleic acid fragment”are used interchangeably herein. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof.

A nucleic acid fragment is “hybridizable” to another nucleic acidfragment, such as a cDNA, genomic DNA, or RNA molecule, when asingle-stranded form of the nucleic acid fragment can anneal to theother nucleic acid fragment under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989),particularly Chapter 11 and Table 11.1 therein (entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments (such ashomologous sequences from distantly related organisms), to highlysimilar fragments (such as genes that duplicate functional enzymes fromclosely related organisms). Post-hybridization washes determinestringency conditions. One set of preferred conditions uses a series ofwashes starting with 6×SSC, 0.5% SDS at room temperature for 15 min,then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and thenrepeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A morepreferred set of stringent conditions uses higher temperatures in whichthe washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C.Another preferred set of highly stringent conditions uses two finalwashes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringentconditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washedwith 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. Preferably a minimum length for a hybridizable nucleic acidis at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least about 30nucleotides. Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)).In general, a sequence of ten or more contiguous amino acids or thirtyor more nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to specifically identify and/or isolatea nucleic acid fragment comprising the sequence. The instantspecification teaches the complete amino acid and nucleotide sequenceencoding one or more particular Δ8 desaturase proteins. The skilledartisan, having the benefit of the sequences as reported herein, may nowuse all or a substantial portion of the disclosed sequences for purposesknown to those skilled in this art. Accordingly, the instant inventioncomprises the complete sequences as reported in the accompanyingSequence Listing, as well as substantial portions of those sequences asdefined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. Accordingly, the invention hereinalso includes isolated nucleic acid fragments that are complementary tothe complete sequences as reported in the accompanying Sequence Listing,as well as those substantially similar nucleic acid sequences.

The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate gene expression or produce a certain phenotype. These termsalso refer to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially alter the functional properties of the resultingnucleic acid fragment relative to the initial, unmodified fragment. Itis therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize, under moderately stringent conditions (e.g.,0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or toany portion of the nucleotide sequences disclosed herein and which arefunctionally equivalent to any of the nucleic acid sequences disclosedherein.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment that encodes all or a substantialportion of the amino acid sequence encoding the instant Δ8 desaturasepolypeptides as set forth in SEQ ID NOs:2, 10 and 12. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

“Chemically synthesized”, as related to a sequence of DNA, means thatthe component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established proceduresor, automated chemical synthesis can be performed using one of a numberof commercially available machines. “Synthetic genes” can be assembledfrom oligonucleotide building blocks that are chemically synthesizedusing procedures known to those skilled in the art. These buildingblocks are ligated and annealed to form gene segments that are thenenzymatically assembled to construct the entire gene. Accordingly, thegenes can be tailored for optimal gene expression based on optimizationof nucleotide sequence to reflect the codon bias of the host cell. Theskilled artisan appreciates the likelihood of successful gene expressionif codon usage is biased towards those codons favored by the host.Determination of preferred codons can be based on a survey of genesderived from the host cell, where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, and that may refer to the coding region alone or may includeregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers any gene that is not a native gene, comprising regulatoryand coding sequences that are not found together in nature. Accordingly,a chimeric gene may comprise regulatory sequences and coding sequencesthat are derived from different sources, or regulatory sequences andcoding sequences derived from the same source, but arranged in a mannerdifferent than that found in nature. “Endogenous gene” refers to anative gene in its natural location in the genome of an organism. A“foreign” gene refers to a gene not normally found in the host organism,but that is introduced into the host organism by gene transfer. Foreigngenes can comprise native genes inserted into a non-native organism,native genes introduced into a new location within the native host, orchimeric genes. A “transgene” is a gene that has been introduced intothe genome by a transformation procedure. A “codon-optimized gene” is agene having its frequency of codon usage designed to mimic the frequencyof preferred codon usage of the host cell.

An “allele” is one of several alternative forms of a gene occupying agiven locus on a chromosome. When all the alleles present at a givenlocus on a chromosome are the same that plant is homozygous at thatlocus. If the alleles present at a given locus on a chromosome differ,that plant is heterozygous at that locus.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to: promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequence mayconsist of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence that can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.It is further recognized that since in most cases the exact boundariesof regulatory sequences have not been completely defined, DNA fragmentsof some variation may have identical promoter activity. Promoters thatcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro, J. K., andGoldberg, R. B. Biochemistry of Plants, 15:1-82 (1989).

“Translation leader sequence” refers to a polynucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D., Mol.Biotechnol., 3:225-236 (1995)).

The terms “3′ non-coding sequences”, “transcription terminator” and“termination sequences” refer to DNA sequences located downstream of acoding sequence. This includes polyadenylation recognition sequences andother sequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The 3′ region can influence thetranscription, RNA processing or stability, or translation of theassociated coding sequence. The use of different 3′ non-coding sequencesis exemplified by Ingelbrecht, I. L., et al. (Plant Cell, 1:671-680(1989)).

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript. A RNA transcript is referred toas the mature RNA when it is a RNA sequence derived frompost-transcriptional processing of the primary transcript. “MessengerRNA” or “mRNA” refers to the RNA that is without introns and that can betranslated into protein by the cell. “cDNA” refers to a DNA that iscomplementary to, and synthesized from, a mRNA template using the enzymereverse transcriptase. The cDNA can be single-stranded or converted intodouble-stranded form using the Klenow fragment of DNA polymerase 1.“Sense” RNA refers to RNA transcript that includes the mRNA and can betranslated into protein within a cell or in vitro. “Antisense RNA”refers to a RNA transcript that is complementary to all or part of atarget primary transcript or mRNA, and that blocks the expression of atarget gene (U.S. Pat. No. 5,107,065; WO 99/28508). The complementarityof an antisense RNA may be with any part of the specific genetranscript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence,introns, or the coding sequence. “Functional RNA” refers to antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes. The terms “complement” and “reversecomplement” are used interchangeably herein with respect to mRNAtranscripts, and are meant to define the antisense RNA of the message.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Expression cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that allow for enhanced expression of that gene in a foreign host.

The terms “recombinant construct”, “expression construct”, “chimericconstruct”, “construct”, and “recombinant DNA construct” are usedinterchangeably herein. A recombinant construct comprises an artificialcombination of nucleic acid fragments, e.g., regulatory and codingsequences that are not found together in nature. For example, a chimericconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. Such a construct may be used byitself or may be used in conjunction with a vector. If a vector is used,then the choice of vector is dependent upon the method that will be usedto transform host cells as is well known to those skilled in the art.For example, a plasmid vector can be used. The skilled artisan is wellaware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleic acid fragments of the invention.The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al.,Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events mustbe screened in order to obtain strains or lines displaying the desiredexpression level and pattern. Such screening may be accomplished bySouthern analysis of DNA blots (Southern, J. Mol. Biol., 98:503 (1975)),Northern analysis of mRNA expression (Kroczek, J. Chromatogr. Biomed.Appl., 618(1-2):133-145 (1993)), Western and/or Elisa analyses ofprotein expression, phenotypic analysis or GC analysis of the PUFAproducts, among others.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragments of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

“Mature” protein refers to a post-translationally processed polypeptide(i.e., one from which any pre- or propeptides present in the primarytranslation-product have been removed). “Precursor” protein refers tothe primary product of translation of mRNA (i.e., with pre- andpropeptides still present). Pre- and propeptides may be, but are notlimited to, intracellular localization signals.

“Transformation” refers to the transfer of a nucleic acid fragment intoa genome of a host organism, including both nuclear and organellargenomes, resulting in genetically stable inheritance. In contrast,“transient transformation” refers to the transfer of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Thus, the nucleic acid molecule used for transformation maybe a plasmid that replicates autonomously, for example, or, it mayintegrate into the genome of the host organism. Host organismscontaining the transformed nucleic acid fragments are referred to as“transgenic” or “recombinant” or “transformed” organisms.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Co-suppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020).Co-suppression constructs in plants previously have been designed byfocusing on overexpression of a nucleic acid sequence having homology toan endogenous mRNA, in the sense orientation, which results in thereduction of all RNA having homology to the overexpressed sequence(Vaucheret et al., Plant J., 16:651-659 (1998); Gura, Nature,404:804-808 (2000)). The overall efficiency of this phenomenon is low,and the extent of the RNA reduction is widely variable. Subsequent workhas described the use of “hairpin” structures that incorporate all, orpart, of a mRNA encoding sequence in a complementary orientation thatresults in a potential “stem-loop” structure for the expressed RNA (WO99/53050; WO 02/00904). This increases the frequency of co-suppressionin the recovered transgenic plants. Another variation describes the useof plant viral sequences to direct the suppression, or “silencing”, ofproximal mRNA encoding sequences (WO 98/36083). Both of theseco-suppressing phenomena have not been elucidated mechanistically,although genetic evidence has begun to unravel this complex situation(Elmayan et al., Plant Cell, 10:1747-1757 (1998)).

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2^(nd) Ed., Plenum, 1980). Generally, the cellular oil orTAG content of these microorganisms follows a sigmoid curve, wherein theconcentration of lipid increases until it reaches a maximum at the latelogarithmic or early stationary growth phase and then graduallydecreases during the late stationary and death phases (Yongmanitchai andWard, Appl. Environ. Microbiol., 57:419-25 (1991)).

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that can make oil. It is not uncommon for oleaginousmicroorganisms to accumulate in excess of about 25% of their dry cellweight as oil. Examples of oleaginous yeast include, but are no meanslimited to, the following genera: Yarrowia, Candida, Rhodotorula,Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991). Preferred methods to determine identity aredesigned to give the best match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs.

The “Clustal V method of alignment” corresponds to the alignment methodlabeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153(1989); Higgins, D. G. et al. (1992) Comput. Appl. Biosci. 8:189-191)and found in the MegAlign™ program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Sequence alignments andpercent identity calculations may be performed using the MegAlign™program. Multiple alignment of the sequences is performed using theClustal method of alignment (Higgins and Sharp, CABIOS, 5:151-153(1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992))with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10), unlessotherwise specified. Default parameters for pairwise alignments andcalculation of percent identity of protein sequences using the Clustal Vmethod are: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.After alignment of the sequences using the Clustal program, it ispossible to obtain a “percent identity” by viewing the “sequencedistances” table in the same program.

The “Clustal W method of alignment” corresponds to the alignment methodlabeled Clustal W (described by Thompson et al., Nucleic Acids Res.22:4673-4680 (1994)) and found in the MegAlign™ v5.07 program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Default parameters for multiple alignments and calculation of percentidentity of protein sequences are GAP PENALTY=10, GAP LENGTHPENALTY=0.2, DELAY DIVERGENCE SEQS(%)=30, DNA TRANSITION WEIGHT=0.50,protein weight matrix=Gonnet series and DNA weight matrix=IUB, unlessotherwise specified. Default parameters for pairwise alignments andcalculation of percent identity of protein sequences are GAP PENALTY=10,GAP LENGTH PENALTY=0.1, protein weight matrix=Gonnet 250 and DNA weightmatrix=IUB, unless otherwise specified.

“BLASTN method of alignment” is an algorithm provided by the NationalCenter for Biotechnology Information (NCBI) to compare nucleotidesequences using default parameters.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or anyinteger percentage from 50% to 100%. Indeed, any integer amino acididentity from 50% to 100% may be useful in describing the presentinvention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Also, ofinterest is any full-length or partial complement of this isolatednucleotide fragment.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: (1) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); (2) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); (3) DNASTAR (DNASTAR Inc., Madison, Wis.); (4)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and (5) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

Suitable nucleic acid fragments (isolated polynucleotides of the presentinvention) encode polypeptides that are at least about 85% identical tothe amino acid sequences reported herein. More preferred nucleic acidfragments encode amino acid sequences that are at least about 90%identical to the amino acid sequences reported herein while mostpreferred are nucleic acid fragments that encode amino acid sequencesthat are at least about 95% identical. It is well understood by oneskilled in the art that many levels of sequence identity are useful inidentifying polypeptides from other species, wherein such polypeptideshave the same or similar function or activity; although preferred rangesare described above, any integer percentage from 85% to 100% is usefulfor the purposes herein.

Suitable nucleic acid fragments not only have the above homologies buttypically encode a polypeptide having at least 50 amino acids,preferably at least 100 amino acids, more preferably at least 150 aminoacids, still more preferably at least 200 amino acids, and mostpreferably at least 250 amino acids.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. andEnquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. etal., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987).

An Overview: Biosynthesis of Omega Fatty Acids and Triacylglycerols

The metabolic process wherein oleic acid is converted to ω-3/ω-6 fattyacids involves elongation of the carbon chain through the addition ofcarbon atoms and desaturation of the molecule through the addition ofdouble bonds. This requires a series of special desaturation andelongation enzymes present in the endoplasmic reticulim membrane.However, as seen in FIG. 1 and as described below, there are oftenmultiple alternate pathways for production of a specific ω-3/ω-6 fattyacid.

Specifically, all pathways require the initial conversion of oleic acidto LA, the first of the ω-6 fatty acids, by a Δ12 desaturase. Then,using the “Δ9 elongase/Δ8 desaturase pathway”, ω-6 fatty acids areformed as follows: (1) LA is converted to EDA by a Δ9 elongase; (2) EDAis converted to DGLA by a Δ8 desaturase; and (3) DGLA is converted toARA by a Δ5 desaturase. Alternatively, the “Δ9 elongase/Δ8 desaturasepathway” can be utilized for formation of ω-3 fatty acids as follows:(1) LA is converted to ALA, the first of the ω-3 fatty acids, by a Δ15desaturase; (2) ALA is converted to ETrA by a Δ9 elongase; (3) ETrA isconverted to ETA by a Δ8 desaturase; (4) ETA is converted to EPA by a Δ5desaturase; (5) EPA is converted to DPA by a C_(20/22) elongase; and (6)DPA is converted to DHA by a Δ4 desaturase. Optionally, ω-6 fatty acidsmay be converted to ω-3 fatty acids; for example, ETA and EPA areproduced from DGLA and ARA, respectively, by Δ17 desaturase activity.

Alternate pathways for the biosynthesis of ω-3/ω-6 fatty acids utilize aΔ6 desaturase and C_(18/20) elongase (i.e., the “Δ6 desaturase/Δ6elongase pathway”). More specifically, LA and ALA may be converted toGLA and STA, respectively, by a Δ6 desaturase; then, a C_(18/20)elongase converts GLA to DGLA and/or STA to ETA.

It is contemplated that the particular functionalities required to beexpressed in a specific host organism for production of ω-3/ω-6 fattyacids will depend on the host cell (and its native PUFA profile and/ordesaturase/elongase profile), the availability of substrate, and thedesired end product(s). For example, expression of the Δ9 elongase/Δ8desaturase pathway may be preferred in some embodiments, as opposed toexpression of the Δ6 desaturase/Δ6 elongase pathway, since PUFAsproduced via the former pathway are devoid of GLA.

One skilled in the art will be able to identify various candidate genesencoding each of the enzymes desired for ω3/ω-6 fatty acid biosynthesis.Useful desaturase and elongase sequences may be derived from any source,e.g., isolated from a natural source (from bacteria, algae, fungi,plants, animals, etc.), produced via a semi-synthetic route orsynthesized de novo. Although the particular source of the desaturaseand elongase genes introduced into the host is not critical,considerations for choosing a specific polypeptide having desaturase orelongase activity include: 1.) the substrate specificity of thepolypeptide; 2.) whether the polypeptide or a component thereof is arate-limiting enzyme; 3.) whether the desaturase or elongase isessential for synthesis of a desired PUFA; and/or 4.) co-factorsrequired by the polypeptide. The expressed polypeptide preferably hasparameters compatible with the biochemical environment of its locationin the host cell (see WO 2004/101757).

In additional embodiments, it will also be useful to consider theconversion efficiency of each particular desaturase and/or elongase.More specifically, since each enzyme rarely functions with 100%efficiency to convert substrate to product, the final lipid profile ofun-purified oils produced in a host cell will typically be a mixture ofvarious PUFAs consisting of the desired ω-3/ω-6 fatty acid, as well asvarious upstream intermediary PUFAs. Thus, consideration of eachenzyme's conversion efficiency is also an important variable whenoptimizing biosynthesis of a desired fatty acid that must be consideredin light of the final desired lipid profile of the product.

With each of the considerations above in mind, candidate genes havingthe appropriate desaturase and elongase activities (e.g., Δ6desaturases, C_(18/20) elongases, Δ5 desaturases, Δ17 desaturases, Δ15desaturases, Δ9 desaturases, Δ12 desaturases, C_(14/16) elongases,C_(16/18) elongases, Δ9 elongases, Δ8 desaturases, Δ4 desaturases andC_(20/22) elongases) can be identified according to publicly availableliterature (e.g., GenBank), the patent literature, and experimentalanalysis of organisms having the ability to produce PUFAs. These geneswill be suitable for introduction into a specific host organism, toenable or enhance the organism's synthesis of PUFAs.

Once fatty acids are synthesized within an organism (including saturatedand unsaturated fatty acids and short-chain and long-chain fatty acids),they may be incorporated into triacylglycerides (TAGs). TAGs (theprimary storage unit for fatty acids, including PUFAs) are formed by aseries of reactions that involve: 1.) the esterification of one moleculeof acyl-CoA to glycerol-3-phosphate via an acyltransferase to producelysophosphatidic acid; 2.) the esterification of a second molecule ofacyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate(commonly identified as phosphatidic acid); 3.) removal of a phosphateby phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and4.) the addition of a third fatty acid by the action of anacyltransferase to form TAG.

Sequence Identification of a Euglena gracilis Δ8 Desaturase

Commonly owned WO 2006/012325 and WO 2006/012326 disclose a E. gracilisΔ8 desaturase able to desaturate EDA and EtrA (identified therein as“Eg5 and assigned SEQ ID NO:2). In the present application, the E.gracilis Δ8 desaturase described as “EgD8” (SEQ ID NOs:11 and 12 herein)is 100% identical and equivalent to the nucleotide and amino acidsequences of Eg5.

As is well known in the art, codon-optimization can be a useful means tofurther optimize the expression of an enzyme in an alternate host, sinceuse of host-preferred codons can substantially enhance the expression ofthe foreign gene encoding the polypeptide. As such, a synthetic Δ8desaturase derived from Euglena gracilis and codon-optimized forexpression in Yarrowia lipolytica was also disclosed in WO 2006/012325and WO 2006/012326 as SEQ ID NOs:112 and 113 (designated therein as“D8SF”). Specifically, 207 bp (16.4%) of the 1263 bp coding region weremodified, corresponding to codon-optimization of 192 codons.Additionally, “D8SF” had one additional valine amino acid insertedbetween amino acid residues 1 and 2 of the wildtype Eg5; thus, the totallength of the codon-optimized desaturase is 422 amino acids. Expressionof the codon-optimized gene (i.e., “D8SF”) in Y. lipolytica demonstratedmore efficient desaturation of EDA to DGLA and/or ETrA to ETA than thewildtype gene (i.e., Eg5). In the present application, the synthetic Δ8desaturase derived from E. gracilis and codon-optimized for expressionin Y. lipolytica described as “EgD8S” (SEQ ID NOs:9 and 10 herein) is100% identical and equivalent to the nucleotide and amino acid sequencesof D8SF.

Engineering Targeted Mutations within the Synthetic Δ8 Desaturase,Derived from Euglena gracilis and Codon-Optimized for Expression inYarrowia lipolytica

Methods for synthesizing sequences and bringing sequences together arewell established in the literature. Many techniques are commonlyemployed in the literature to obtain mutations of naturally occurringdesaturase genes (wherein such mutations may include deletions,insertions and point mutations, or combinations thereof). This wouldpermit production of a polypeptide having desaturase activity,respectively, in vivo with more desirable physical and kineticparameters for function in the host cell such as a longer half-life or ahigher rate of production of a desired PUFA. Or, if desired, the regionsof a polypeptide of interest (i.e., a desaturase) important forenzymatic activity can be determined through routine mutagenesis,expression of the resulting mutant polypeptides and determination oftheir activities. All such mutant proteins and nucleotide sequencesencoding them that are derived from the wildtype (i.e., SEQ ID NOs:11and 12) and synthetic codon-optimized (SEQ ID NOs:9 and 10) Δ8desaturase described supra are within the scope of the presentinvention.

More specifically in the invention herein, mutant sequences encoding Δ8desaturases were synthetically engineered, by making targeted mutationswithin the known, functional Euglena gracilis Δ8 desaturase that wascodon-optimized for expression in Yarrowia lipolytica (i.e., “EgD8S”, asset forth in SEQ ID NOs:9 and 10). The effect of each mutation on the Δ8desaturase activity of the resulting mutant EgD8S was screened. Althoughnot to be construed as limiting to the invention herein, a mutant EgD8Senzyme (SEQ ID NO:2) was ultimately created comprising at least oneamino acid mutation (and up to about 33 amino acid mutations) withrespect to the synthetic codon-optimized EgD8S and having functionallyequivalent Δ8 desaturase activity, using the methodology describedbelow.

Creation of a Topological Model and Identification of Suitable AminoAcid Sites for Mutation

General characteristics of Δ8 desaturases, based on desaturaseevolution, are well-described by P. Sperling et al. (ProstaglandinsLeukot. Essent. Fatty Acids, 68:73-95 (2003)). Along with Δ6, Δ5 and Δ4desaturases, Δ8 desaturases are known as long-chain PUFA “front-end”desaturases (wherein desaturation occurs between a pre-existing doublebond and the carboxyl terminus of the fatty acid's acyl group, asopposed to methyl-directed desaturation). These desaturases arecharacterized by three histidine boxes [H(X)₃₋₄H (SEQ ID NOs:166 and167), H(X)₂₋₃HH (SEQ ID NOs:168 and 169) and H/Q(X)₂₋₃HH (SEQ ID NOs:170and 171)] and are members of the cytochrome b₅ fusion superfamily, sincethey possess a fused cytochrome b₅ domain at their N-terminus whichserves as an electron donor. The cytochrome b₅ domain also contains anabsolutely conserved heme-binding motif (i.e., “HPGG”) which has beenshown to be necessary for enzyme activity (J. A. Napier, et al.,Prostaglandins Leukot. Essent. Fatty Acids, 68:135-143 (2003); P.Sperling, et al., supra). Finally, although the crystal structure of a“front-end” desaturase is not yet available, hydropathy plots reveal 4-6membrane spanning helices that account for nearly 30% of the amino acidsequence of these proteins.

Based on the generalizations above, the protein sequence of EgD8S (SEQID NO:10) was specifically analyzed to enable creation of a topologicalmodel (FIG. 2). As expected, EgD8S contained two domains: an N-terminalcytochrome b₅ domain (located between amino acid residues 5 to 71 of SEQID NO:10) and a C-terminal desaturase domain (located between amino acidresidues 79 to 406 of SEQ ID NO:10). Four membrane-spanning helices wereidentified at amino acid residues 88-109, 113-132, 266-283 and 287-309of SEQ ID NO:10 (labeled as regions I, II, III and IV on FIG. 2), withboth the N- and C-termini located on the cytoplasmic side of themembrane. Two additional hydrophobic regions were located at amino acidresidues 157-172 and 223-245. Finally, the three histidine boxes werelocated between amino acid residues 146-150, 183-187 and 358-362, andthe conserved heme-binding motif (“HPGG”) was located at amino acidresidues 27-30.

Using the topological model, alignment of EgD8S with other front-enddesaturases and alignment of EgD8S's cytochrome b₅ domain with othercytochrome b₅ genes, 70 sites within EgD8S were subsequently selected aspossibly suitable for mutagenesis (criteria for selection are describedin detail in Example 2). These sites corresponded to 126 individualamino acid mutations (i.e., 57.9% conserved amino acid substitutions and42.1% non-conserved amino acid substitutions), as detailed in Table 7 ofExample 2.

Site-Directed Mutagenesis for Creation of EgD8S Mutants

Although a variety of approaches may be used for mutagenesis of a Δ8desaturase enzyme, based on the strategies herein it was desirable tocreate specific point mutations within EgD8S usingoligonucleotide-mediated site-directed mutagenesis. Furthermore,although numerous site-directed mutagenesis protocols exist (e.g.,Ishii, T. M., et al., Methods Enzymol., 293:53-71 (1998); Ling M. M. andB. H. Robinson, Anal. Biochem., 254:157-178 (1997); Braman J. (ed.) InVitro Mutagenesis Protocols. 2^(nd) Ed., Humania: Totowa, N.J. (2002);Kunkel T. A., et al., Methods Enzymol., 154:367-382 (1987); Sawano A.and Miyawaki, A. Nucleic Acids Res., 28:e78 (2000)), the QuikChange®site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) wasselected for use based on its facile implementation and high efficiency.Specifically, the kit requires no specialized vectors, uniquerestriction sites, or multiple transformations and allows site-specificmutation in virtually any double-stranded plasmid. The basic procedureutilizes a supercoiled double-stranded DNA vector with an insert ofinterest and two synthetic oligonucleotide primers containing thedesired mutation. The oligonucleotide primers, each complementary toopposite strands of the vector, are extended during temperature cyclingby a DNA polymerase. Incorporation of the oligonucleotide primersgenerates a mutated plasmid containing staggered nicks. Followingtemperature cycling, the product is treated with Dpn I endonuclease(specific for methylated and hemi-methylated DNA) as a means to digestthe parental DNA template and to select for newly synthesized mutantDNA. The nicked vector DNA containing the desired mutations is thentransformed and propagated in an Escherichia coli host.

Using the techniques described above, the feasibility of engineering asynthetic EgD8S (having multiple point mutations with respect to EgD8Sbut maintaining functional equivalence with respect to the enzyme's Δ8desaturase activity) was then tested. Specifically, selected individualpoint mutations were introduced by site-directed mutagenesis into EgD8S(within a plasmid construct comprising a chimeric FBAINm::EgD8S::XPRgene), transformed into E. coli, and then screened for Δ8 desaturaseactivity based on GC analyses.

The skilled person will be able to envision additional screens for theselection of genes encoding proteins having Δ8 desaturase activity. Forexample, desaturase activity may be demonstrated by assays in which apreparation containing an enzyme is incubated with a suitable form ofsubstrate fatty acid and analyzed for conversion of this substrate tothe predicted fatty acid product. Alternatively, a DNA sequence proposedto encode a desaturase protein may be incorporated into a suitablevector construct and thereby expressed in cells of a type that do notnormally have an ability to desaturate a particular fatty acidsubstrate. Activity of the desaturase enzyme encoded by the DNA sequencecan then be demonstrated by supplying a suitable form of substrate fattyacid to cells transformed with a vector containing thedesaturase-encoding DNA sequence and to suitable control cells (e.g.,transformed with the empty vector alone). In such an experiment,detection of the predicted fatty acid product in cells containing thedesaturase-encoding DNA sequence and not in control cells establishesthe desaturase activity.

Results from the experiment described above resulted in theidentification of some mutations that resulted in completelynon-functional mutant Δ8 desaturases having 0% Δ8 desaturase activity(e.g., simultaneous mutation of 48V to F and 49M to L or simultaneousmutation of 304G to F and 305F to G). Despite this, ˜75% of theindividual mutations tested did not significantly diminish the mutantenzyme's Δ8 desaturase activity as compared to the Δ8 desaturaseactivity of EgD8S. More specifically, the following mutations wereidentified as preferred mutations, wherein Δ8 desaturase activity wasfunctionally equivalent (i.e., 100%) or greater than that of EgD8S(i.e., SEQ ID NO:10):

TABLE 4 Initial Preferred Mutations Within EgD8S Sequence Mutations WithMutation Respect to EgD8S % Site (SEQ ID NO: 10) Activity* M3 16T to K,17T to V 100% M8 66P to Q, 67S to A 100% M12 407A to S, 408V to Q 100%M14 416Q to V, 417P to Y 100% M16 108S to L 100% M19 122V to S 100% M3854A to G, 55F to Y 100% M39 64I to L, 65N to D 100% M40 69E to D, 70L toV 100% M41 75A to G, 76V to L 100% M45 117G to A, 118Y to F 100% M46132V to L, 133L to V 100% M49 297F to V, 298V to L 100% M50 309I to V,310V to I 100% M51 347I to L, 348T to S 100% M51B 346I to V, 347I to L,348T to S 100% M53 9L to V 100% M54 19D to E, 20V to I 100% M58 65N to Q100% M63 279T to L, 280L to T 100% M68 162L to V, 163V to L 100% M69170G to A, 171L to V 100% M70 418A to G, 419G to A 100% M2 12T to V 110%M22 127Y to Q 110% M26 293L to M 110% M6 59K to L 110% M1 4S to A, 5K toS 115% M21 125Q to H, 126M to L 120% M15 422L to Q 125% *“% Activity”refers to the Δ8 desaturase activity of each mutant EgD8S with respectto the Δ8 desaturase activity of EgD8S, as set forth as SEQ ID N0: 10.

It will be appreciated by one of skill in the art that the useful mutantΔ8 desaturases of the present invention are not limited to the 30mutation combinations described above. For example, although themutation site described as M3 includes two specific amino acid mutations(i.e., 16T to K and 17T to V), one skilled in the art will expect that asingle mutation of either 16T to K or 17T to V will have utility in thedesign of a mutant Δ8 desaturase whose Δ8 desaturase activity is atleast about functionally equivalent to the Δ8 desaturase activity of thesynthetic codon-optimized EgD8S. Thus, in actuality, Table 4 presents 52single amino acid mutations that are useful for the purposes herein, inthe design of a mutant Δ8 desaturase having Δ8 desaturase activity thatis at least about functionally equivalent to the Δ8 desaturase activityof SEQ ID NO:10.

Based on the results above, experimental work was continued in an effortto “stack” appropriate individual amino acid mutations within thesynthetic codon-optimized EgD8S sequence. This resulted in creation of amutant Δ8 desaturase as set forth in SEQ ID NO:2 having “n” amino acidmutations, wherein “n” is any integer from 1 to 33 inclusive (i.e., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33), and having Δ8desaturase activity comparable to that of EgD8S. Specifically, SEQ IDNO:2 comprises at least one mutation selected from the group consistingof: 4S to A, 5K to S, 12T to V, 16T to K, 17T to V, 54A to G, 55F to Y,66P to Q, 67S to A, 108S to L, 117G to A, 118Y to F, 120L to M, 121M toL, 125Q to H, 126M to L, 132V to L, 133L to V, 162L to V, 163V to L,170G to A, 171L to V, 279T to L, 280L to T, 293L to M, 3461 to V, 3471to L, 348T to S, 407A to S, 408V to Q, 418A to G, 419G to A and 422L toQ, wherein the mutations are defined with respect to the syntheticcodon-optimized sequence of EgD8S (i.e., SEQ ID NO:10); wherein SEQ IDNO:2 is not 100% identical to SEQ ID NO:10; and wherein the mutant EgD8Sis at least about functionally equivalent to EgD8S (SEQ ID NO:10). Itwill be appreciated by the skilled person that each of the abovemutations can be used in any combination with one another. And, all suchmutant proteins and nucleotide sequences encoding them that are derivedfrom EgD8 and/or EgD8S as described herein are within the scope of thepresent invention. In more preferred embodiments, the mutant EgD8S hasat least about 10-18 conservative and non-conservative amino acidsubstitutions (i.e., mutations) with respect to the syntheticcodon-optimized sequence of EgD8S, more preferably at least about 19-25conservative and non-conservative amino acid substitutions, and mostpreferably at least about 26-33 conservative and non-conservative aminoacid substitutions with respect to the synthetic codon-optimizedsequence of EgD8S (i.e., SEQ ID NO:10). Thus, for example, in onepreferred embodiment mutant EgD8S-23 (SEQ ID NO:4) comprises thefollowing 24 amino acid mutations with respect to the syntheticcodon-optimized EgD8S sequence set forth as SEQ ID NO:10: 4S to A, 5K toS, 12T to V, 16T to K, 17T to V, 66P to Q, 67S to A, 108S to L, 117G toA, 118Y to F, 120L to M, 121M to L, 125Q to H, 126M to L, 132V to L, 133L to V, 162L to V, 163V to L, 293L to M, 407A to S, 408V to Q, 418A toG, 419G to A and 422L to Q. Pairwise alignment of the mutant EgD8S-23amino acid sequence to the synthetic codon-optimized sequence of SEQ IDNO:10 using default parameters of Vector NTI®'s AlignX program(Invitrogen Corporation, Carlsbad, Calif.) revealed 94.3% sequenceidentity and 97.9% consensus between the two proteins over a length of422 amino acids.

In another preferred embodiment, mutant EgD8S-013 (SEQ ID NO:6)comprises the following 28 amino acid mutations with respect to thesynthetic codon-optimized EgD8S sequence set forth as SEQ ID NO:10: 4Sto A, 5K to S, 12T to V, 16T to K, 17T to V, 54A to G, 55F to Y, 66P toQ, 67S to A, 108S to L, 117G to A, 118Y to F, 120L to M, 121M to L, 125Qto H, 126M to L, 132V to L, 133L to V, 162L to V, 163V to L, 170G to A,171L to V, 293L to M, 407A to S, 408V to Q, 418A to G, 419G to A and422L to Q. Pairwise alignment of the mutant EgD8S-013 amino acidsequence to the synthetic codon-optimized sequence of SEQ ID NO:10 usingdefault parameters of Vector NTI®'s AlignX program revealed 93.4%sequence identity and 97.9% consensus between the two proteins over alength of 422 amino acids.

In another preferred embodiment, mutant EgD8S-015 (SEQ ID NO:8)comprises the following 31 amino acid mutations with respect to thesynthetic codon-optimized EgD8S sequence set forth as SEQ ID NO:10: 4Sto A, 5K to S, 12T to V, 16T to K, 17T to V, 54A to G, 55F to Y, 66P toQ, 67S to A, 108S to L, 117G to A, 118Y to F, 120L to M, 121M to L, 125Qto H, 126M to L, 162L to V, 163V to L, 170G to A, 171L to V, 293L to M,279T to L, 280L to T, 3461 to V, 3471 to L, 348T to S, 407A to S, 408Vto Q, 418A to G, 419G to A and 422L to Q. Pairwise alignment of themutant EgD8S-015 amino acid sequence to the synthetic codon-optimizedsequence of SEQ ID NO:10 using default parameters of Vector NTI®'sAlignX program revealed 92.7% sequence identity and 97.4% consensusbetween the two proteins over a length of 422 amino acids.

Thus, in one embodiment, the present invention concerns an isolatedpolynucleotide comprising:

-   -   (a) a nucleotide sequence encoding a mutant polypeptide having        Δ8 desaturase activity, wherein the mutant polypeptide has an        amino acid sequence as set forth in SEQ ID NO:2, wherein:        -   (i) SEQ ID NO:2 comprises at least one mutation selected            from the group consisting of: 4S to A, 5K to S, 12T to V,            16T to K, 17T to V, 54A to G, 55F to Y, 66P to Q, 67S to A,            108S to L, 117G to A, 118Y to F, 120L to M, 121M to L, 125Q            to H, 126M to L, 132V to L, 133L to V, 162L to V, 163V to L,            170G to A, 171L to V, 279T to L, 280L to T, 293L to M, 3461            to V, 3471 to L, 348T to S, 407A to S, 408V to Q, 418A to G,            419G to A and 422L to Q, wherein the mutations are defined            with respect to the synthetic codon-optimized sequence of            EgD8S (i.e., SEQ ID NO:10); and,        -   (ii) SEQ ID NO:2 is not identical to SEQ ID NO:10; or,    -   (b) a complement of the nucleotide sequence, wherein the        complement and the nucleotide sequence consist of the same        number of nucleotides and are 100% complementary.        In further preferred embodiments, the Δ8 desaturase activity of        SEQ ID NO:2, as described above, is at least about functionally        equivalent to the Δ8 desaturase activity of SEQ ID NO:10.

Alternate Means of Mutagenesis for Creation of EgD8S Mutants

As is well known to one of skill in the art, in vitro mutagenesis andselection or error prone PCR (Leung et al., Techniques, 1:11-15 (1989);Zhou et al., Nucleic Acids Res., 19:6052-6052 (1991); Spee et al.,Nucleic Acids Res., 21:777-778 (1993); Melnikov et al., Nucleic AcidsRes., 27(4):1056-1062 (Feb. 15, 1999)) could also be employed as a meansto obtain mutations of naturally occurring desaturase genes, such asEgD8 or EgD8S, wherein the mutations may include deletions, insertionsand point mutations, or combinations thereof. The principal advantage oferror-prone PCR is that all mutations introduced by this method will bewithin the desired desaturase gene, and any change may be easilycontrolled by changing the PCR conditions. Alternatively, in vivomutagenesis may be employed using commercially available materials suchas the E. coli XL1-Red strain and Epicurian coli XL1-Red mutator strainfrom Stratagene (La Jolla, Calif.; Greener and Callahan, Strategies,7:32-34 (1994)). This strain is deficient in three of the primary DNArepair pathways (mutS, mutD and mutT), resulting in a mutation rate5000-fold higher than that of wild-type. In vivo mutagenesis does notdepend on ligation efficiency (as with error-prone PCR); however, amutation may occur at any region of the vector and the mutation ratesare generally much lower.

In other embodiments, it is contemplated that a mutant Δ8 desaturaseenzyme with altered or enhanced Δ8 desaturase activity may beconstructed using the method of “gene shuffling” (U.S. Pat. No.5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No. 5,830,721; and U.S.Pat. No. 5,837,458). The method of gene shuffling is particularlyattractive due to its facile implementation and high rate ofmutagenesis. The process of gene shuffling involves the restriction of agene of interest into fragments of specific size in the presence ofadditional populations of DNA regions of both similarity to (ordifference to) the gene of interest. This pool of fragments will thendenature and reanneal to create a mutated gene. The mutated gene is thenscreened for altered activity.

Delta-8 desaturase sequences (i.e., wildtype, synthetic, codon-optimizedor mutant) may be mutated and screened for altered or enhanced Δ8desaturase activity by this method. The sequences should bedouble-stranded and can be of various lengths ranging from 50 bp to 10kB. The sequences may be randomly digested into fragments ranging fromabout 10 bp to 1000 bp, using restriction endonucleases well known inthe art (Maniatis, supra). In addition to the full-length sequences,populations of fragments that are hybridizable to all or portions of thesequence may be added. Similarly, a population of fragments that are nothybridizable to the wildtype sequence may also be added. Typically,these additional fragment populations are added in about a 10- to20-fold excess by weight as compared to the total nucleic acid.Generally this process will allow generation of about 100 to 1000different specific nucleic acid fragments in the mixture. The mixedpopulation of random nucleic acid fragments are denatured to formsingle-stranded nucleic acid fragments and then reannealed. Only thosesingle-stranded nucleic acid fragments having regions of homology withother single-stranded nucleic acid fragments will reanneal. The randomnucleic acid fragments may be denatured by heating. One skilled in theart could determine the conditions necessary to completely denature thedouble-stranded nucleic acid, wherein the temperature is preferably fromabout 80° C. to about 100° C. The nucleic acid fragments may bereannealed by cooling, wherein the temperature is preferably from about20° C. to about 75° C. Renaturation can be accelerated by the additionof polyethylene glycol (“PEG”) or salt, wherein the salt concentrationis preferably from about 0 mM to about 200 mM. The annealed nucleic acidfragments are next incubated in the presence of a nucleic acidpolymerase and dNTP's (i.e., dATP, dCTP, dGTP and dTTP). The nucleicacid polymerase may be the Klenow fragment, the Taq polymerase or anyother DNA polymerase known, in the art. The polymerase may be added tothe random nucleic acid fragments prior to annealing, simultaneouslywith annealing or after annealing. The cycle of denaturation,renaturation and incubation in the presence of polymerase is repeatedfor a desired number of times. Preferably the cycle is repeated from 2to 50 times, and more preferably the sequence is repeated from 10 to 40times. The resulting nucleic acid is a larger double-strandedpolynucleotide of from about 50 bp to about 100 kB and may be screenedfor expression and altered Δ8 desaturase activity by standard cloningand expression protocols (Maniatis, supra).

Irrespective of the method of mutagenesis, it is contemplated that amutant Δ8 desaturase having Δ8 desaturase activity at least aboutfunctionally equivalent to that of EgD8 (SEQ ID NO:12) or EgD8S (SEQ IDNO:10) may be evolved as set forth in SEQ ID NO:2, wherein: (i) SEQ IDNO:2 comprises at least one mutation selected from the group consistingof: 4S to A, 5K to S, 12T to V, 16T to K, 17T to V, 54A to G, 55F to Y,66P to Q, 67S to A, 108S to L, 117G to A, 118Y to F, 120L to M, 121M toL, 125Q to H, 126M to L, 132V to L, 133L to V, 162L to V, 163V to L,170G to A, 171 L to V, 279T to L, 280L to T, 293L to M, 3461 to V, 3471to L, 348T to S, 407A to S, 408V to Q, 418A to G, 419G to A and 422L toQ, wherein the mutations are defined with respect to the syntheticcodon-optimized sequence of EgD8S (i.e., SEQ ID NO:10); and, (ii) SEQ IDNO:2 is not 100% identical to SEQ ID NO:10. Furthermore, it will beappreciated that the invention encompasses not only the specificmutations described above, but also those that allow for thesubstitution of chemically equivalent amino acids. So, for example,where a substitution of an amino acid with the aliphatic, nonpolar aminoacid alanine is made, it will be expected that the same site may besubstituted with the chemically equivalent amino acid serine.

In other embodiments, any of the Δ8 desaturase nucleic acid fragmentsdescribed herein may be used for creation of new and improved fatty aciddesaturases by domain swapping, wherein a functional domain from any ofthe Δ8 desaturase nucleic acid fragments is exchanged with a functionaldomain in an alternate desaturase gene to thereby result in a novelprotein.

Identification and Isolation of Homologs

Any of the instant desaturase sequences (i.e., those mutants derivedfrom EgD8 or EgD8S) or portions thereof may be used to search for Δ8desaturase homologs in the same or other bacterial, algal, fungal,Oomycete or plant species using sequence analysis software. In general,such computer software matches similar sequences by assigning degrees ofhomology to various substitutions, deletions and other modifications.

Alternatively, any of the instant desaturase sequences or portionsthereof may also be employed as hybridization reagents for theidentification of Δ8 homologs. The basic components of a nucleic acidhybridization test include a probe, a sample suspected of containing thegene or gene fragment of interest and a specific hybridization method.Probes of the present invention are typically single-stranded nucleicacid sequences that are complementary to the nucleic acid sequences tobe detected. Probes are “hybridizable” to the nucleic acid sequence tobe detected. Although the probe length can vary from 5 bases to tens ofthousands of bases, typically a probe length of about 15 bases to about30 bases is suitable. Only part of the probe molecule need becomplementary to the nucleic acid sequence to be detected. In addition,the complementarity between the probe and the target sequence need notbe perfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions that will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed. Optionally, achaotropic agent may be added (e.g., guanidinium chloride, guanidiniumthiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodiumperchlorate, rubidium tetrachloroacetate, potassium iodide, cesiumtrifluoroacetate). If desired, one can add formamide to thehybridization mixture, typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 Mbuffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), orbetween 0.5-20 mM EDTA, FICOLL (Pharmacia, Inc.) (about 300-500 kdal),polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g.,calf thymus or salmon sperm DNA, or yeast RNA), and optionally fromabout 0.5 to 2% wt/vol glycine. Other additives may also be included,such as volume exclusion agents that include a variety of polarwater-soluble or swellable agents (e.g., polyethylene glycol), anionicpolymers (e.g., polyacrylate or polymethylacrylate) and anionicsaccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the sequence.

In additional embodiments, any of the Δ8 desaturase nucleic acidfragments described herein (or any homologs identified thereof) may beused to isolate genes encoding homologous proteins from the same orother bacterial, algal, fungal, Oomycete or plant species. Isolation ofhomologous genes using sequence-dependent protocols is well known in theart. Examples of sequence-dependent protocols include, but are notlimited to: 1.) methods of nucleic acid hybridization; 2.) methods ofDNA and RNA amplification, as exemplified by various uses of nucleicacid amplification technologies [e.g., polymerase chain reaction (PCR),Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR),Tabor, S. et al., Proc. Acad. Sci. USA, 82:1074 (1985); or stranddisplacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci.USA, 89:392 (1992)]; and 3.) methods of library construction andscreening by complementation.

For example, genes encoding similar proteins or polypeptides to the Δ8desaturases described herein could be isolated directly by using all ora portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from e.g., any desired marine euglenoid,yeast or fungus using methodology well known to those skilled in the art(wherein those organisms producing EDA or ETrA [or derivatives thereof]would be preferred). Specific oligonucleotide probes based upon theinstant nucleic acid sequences can be designed and synthesized bymethods known in the art (Maniatis, supra). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan (e.g., random primers DNA labeling, nicktranslation or end-labeling techniques), or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part of (or full-length of) the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full-length DNA fragments under conditionsof appropriate stringency.

Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in MolecularBiology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols:Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the instant sequences may be used in PCRprotocols to amplify longer nucleic acid fragments encoding homologousgenes from DNA or RNA. PCR may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding eukaryotic genes.

Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al., PNAS USA, 85:8998 (1988)) togenerate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (Gibco/BRL,Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated(Ohara et al., PNAS USA, 86:5673 (1989); Loh et al., Science, 243:217(1989)).

Methods for Production of Various ω-3 and/or ω-6 Fatty Acids

It is expected that introduction of chimeric genes encoding the Δ8desaturases described herein, under the control of the appropriatepromoters will result in increased production of DGLA and/or ETA in thetransformed host organism, respectively. As such, the present inventionencompasses a method for the direct production of PUFAs comprisingexposing a fatty acid substrate (i.e., EDA or ETrA) to the desaturaseenzymes described herein (i.e., those mutants derived from EgD8 orEgD8S, or homologs thereof), such that the substrate is converted to thedesired fatty acid product (i.e., DGLA and/or ETA).

More specifically, it is an object of the present invention to provide amethod for the production of DGLA in a host cell (e.g., oleaginousyeast, soybean), wherein the host cell comprises:

-   -   a.) a recombinant construct encoding a Δ8 desaturase polypeptide        as set forth in SEQ ID NO:2 wherein SEQ ID NO:2 is not 100%        identical to SEQ ID NO:10; and,    -   b.) a source of EDA;        wherein the host cell is grown under conditions such that the Δ8        desaturase is expressed and the EDA is converted to DGLA, and        wherein the DGLA is optionally recovered.

The person of skill in the art will recognize that the broad substraterange of the Δ8 desaturase may additionally allow for the use of theenzyme for the conversion of ETrA to ETA. Accordingly the inventionprovides a method for the production of ETA, wherein the host cellcomprises:

-   -   a.) a recombinant construct encoding a Δ8 desaturase polypeptide        as set forth in SEQ ID NO:2 wherein SEQ ID NO:2 is not 100%        identical to SEQ ID NO:10; and,    -   b.) a source of ETrA;        wherein the host cell is grown under conditions such that the Δ8        desaturase is expressed and the ETrA is converted to ETA, and        wherein the ETA is optionally recovered.

Alternatively, each Δ8 desaturase gene and its corresponding enzymeproduct described herein can be used indirectly for the production ofω-3 fatty acids (see WO 2004/101757). Indirect production of ω-3/ω-6PUFAs occurs wherein the fatty acid substrate is converted indirectlyinto the desired fatty acid product, via means of an intermediatestep(s) or pathway intermediate(s). Thus, it is contemplated that the Δ8desaturases described herein (i.e., those mutants derived from EgD8 orEgD8S, or homologs thereof) may be expressed in conjunction withadditional genes encoding enzymes of the PUFA biosynthetic pathway(e.g., Δ6 desaturases, C_(18/20) elongases, Δ17 desaturases, Δ15desaturases, Δ9 desaturases, Δ12 desaturases, C_(14/16) elongases,C_(16/18) elongases, Δ9 elongases, Δ5 desaturases, Δ4 desaturases,C_(20/22) elongases) to result in higher levels of production oflonger-chain ω-3/ω-6 fatty acids (e.g., ARA, EPA, DPA and DHA). Inpreferred embodiments, the Δ8 desaturases of the present invention willminimally be expressed in conjunction with a Δ9 elongase (e.g., a Δ9elongase as set forth in SEQ ID NO:173 or SEQ ID NO:176). However, theparticular genes included within a particular expression cassette willdepend on the host cell (and its PUFA profile and/or desaturase/elongaseprofile), the availability of substrate and the desired end product(s).

Plant Expression Systems, Cassettes & Vectors, and Transformation

In one embodiment, this invention concerns a recombinant constructcomprising any one of the Δ8 desaturase polynucleotides of the inventionoperably linked to at least one regulatory sequence suitable forexpression in a plant. A promoter is a DNA sequence that directs thecellular machinery of a plant to produce RNA from the contiguous codingsequence downstream (3′) of the promoter. The promoter region influencesthe rate, developmental stage, and cell type in which the RNA transcriptof the gene is made. The RNA transcript is processed to produce mRNAwhich serves as a template for translation of the RNA sequence into theamino acid sequence of the encoded polypeptide. The 5′ non-translatedleader sequence is a region of the mRNA upstream of the protein codingregion that may play a role in initiation and translation of the mRNA.The 3′ transcription termination/polyadenylation signal is anon-translated region downstream of the protein coding region thatfunctions in the plant cell to cause termination of the RNA transcriptand the addition of polyadenylate nucleotides to the 3′ end of the RNA.

The origin of the promoter chosen to drive expression of the Δ8desaturase coding sequence is not important as long as it has sufficienttranscriptional activity to accomplish the invention by expressingtranslatable mRNA for the desired nucleic acid fragments in the desiredhost tissue at the right time. Either heterologous or non-heterologous(i.e., endogenous) promoters can be used to practice the invention. Forexample, suitable promoters include, but are not limited to: the α-primesubunit of β-conglycinin promoter, the Kunitz trypsin inhibitor 3promoter, the annexin promoter, the Gly1 promoter, the β subunit ofβ-conglycinin promoter, the P34/Gly Bd m 30K promoter, the albuminpromoter, the Leg A1 promoter and the Leg A2 promoter.

The annexin, or P34, promoter is described in WO 2004/071178 (publishedAug. 26, 2004). The level of activity of the annexin promoter iscomparable to that of many known strong promoters, such as: (1) the CaMV35S promoter (Atanassova et al., Plant Mol. Biol., 37:275-285 (1998);Battraw and Hall, Plant Mol. Biol., 15:527-538 (1990); Holtorf et al.,Plant Mol. Biol., 29:637-646 (1995); Jefferson et al., EMBO J.,6:3901-3907 (1987); Wilmink et al., Plant Mol. Biol., 28:949-955(1995)); (2) the Arabidopsis oleosin promoters (Plant et al., Plant Mol.Biol., 25:193-205 (1994); Li, Texas A&M University Ph.D. dissertation,pp. 107-128 (1997)); (3) the Arabidopsis ubiquitin extension proteinpromoters (Callis et al., J. Biol. Chem., 265(21):12486-93 (1990)); (4)a tomato ubiquitin gene promoter (Rollfinke et al., Gene, 211(2):267-76(1998)); (5) a soybean heat shock protein promoter (Schoffl et al., Mol.Gen. Genet., 217(2-3):246-53 (1989)); and, (6) a maize H3 histone genepromoter (Atanassova et al., Plant Mol. Biol., 37(2):275-85 (1989)).

Another useful feature of the annexin promoter is its expression profilein developing seeds. The annexin promoter is most active in developingseeds at early stages (before 10 days after pollination) and is largelyquiescent in later stages. The expression profile of the annexinpromoter is different from that of many seed-specific promoters, e.g.,seed storage protein promoters, which often provide highest activity inlater stages of development (Chen et al., Dev. Genet., 10:112-122(1989); Ellerstrom et al., Plant Mol. Biol., 32:1019-1027 (1996); Keddieet al., Plant Mol. Biol., 24:327-340 (1994); Plant et al., (supra); Li,(supra)). The annexin promoter has a more conventional expressionprofile but remains distinct from other known seed specific promoters.Thus, the annexin promoter will be a very attractive candidate whenoverexpression, or suppression, of a gene in embryos is desired at anearly developing stage. For example, it may be desirable to overexpressa gene regulating early embryo development or a gene involved in themetabolism prior to seed maturation.

Following identification of an appropriate promoter suitable forexpression of a specific Δ8 desaturase coding sequence, the promoter isthen operably linked in a sense orientation using conventional meanswell known to those skilled in the art.

Once the recombinant construct has been made, it may then be introducedinto a plant cell of choice by methods well known to those of ordinaryskill in the art (e.g., transfection, transformation andelectroporation). Oilseed plant cells are the preferred plant cells. Thetransformed plant cell is then cultured and regenerated under suitableconditions permitting expression of the long-chain PUFA which is thenoptionally recovered and purified.

The recombinant constructs of the invention may be introduced into oneplant cell; or, alternatively, each construct may be introduced intoseparate plant cells.

Expression in a plant cell may be accomplished in a transient or stablefashion as is described above.

The desired long-chain PUFAs can be expressed in seed. Also within thescope of this invention are seeds or plant parts obtained from suchtransformed plants.

Plant parts include differentiated and undifferentiated tissuesincluding, but not limited to: roots, stems, shoots, leaves, pollen,seeds, tumor tissue and various forms of cells and culture (e.g., singlecells, protoplasts, embryos and callus tissue). The plant tissue may bein plant or in a plant organ, tissue or cell culture.

The term “plant organ” refers to plant tissue or a group of tissues thatconstitute a morphologically and functionally distinct part of a plant.The term “genome” refers to the following: 1.) the entire complement ofgenetic material (genes and non-coding sequences) that is present ineach cell of an organism, or virus or organelle; and/or 2.) a completeset of chromosomes inherited as a (haploid) unit from one parent.

Thus, this invention also concerns a method for transforming a cell,comprising transforming a cell with a recombinant construct of theinvention and selecting those cells transformed with the recombinantconstruct, wherein: said recombinant construct comprises a nucleotidesequence encoding a mutant polypeptide having Δ8 desaturase activity,wherein the amino acid sequence of the mutant polypeptide is set forthin SEQ ID NO:2, and wherein SEQ ID NO:2 is not identical to SEQ IDNO:10.

Also of interest is a method for producing a transformed plantcomprising transforming a plant cell with the mutant Δ8 desaturasepolynucleotides of the instant invention and regenerating a plant fromthe transformed plant cell.

Methods for transforming dicots (primarily by use of Agrobacteriumtumefaciens) and obtaining transgenic plants have been published, amongothers, for: cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135);soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011); Brassica(U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant Cell Rep.,15:653-657 (1996); McKently et al., Plant Cell Rep., 14:699-703 (1995));papaya (Ling, K. et al., Bio/technology, 9:752-758 (1991)); and pea(Grant et al., Plant Cell Rep., 15:254-258 (1995)). For a review ofother commonly used methods of plant transformation see Newell, C. A.(Mol. Biotechnol., 16:53-65 (2000)). One of these methods oftransformation uses Agrobacterium rhizogenes (Tepfler, M. andCasse-Delbart, F., Microbiol. Sci., 4:24-28 (1987)). Transformation ofsoybeans using direct delivery of DNA has been published using PEGfusion (WO 92/17598), electroporation (Chowrira, G. M. et al., Mol.Biotechnol., 3:17-23 (1995); Christou, P. et al., Proc. Natl. Acad. Sci.Sci. USA, 84:3962-3966 (1987)), microinjection, or particle bombardment(McCabe, D. E. et al., Bio/Technology, 6:923 (1988); Christou et al.,Plant Physiol., 87:671-674 (1988)).

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, In: Methodsfor Plant Molecular Biology, (Eds.), Academic: San Diego, Calif.(1988)). This regeneration and growth process typically includes thesteps of selection of transformed cells and culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for: the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.);generation of recombinant DNA fragments and recombinant expressionconstructs; and, the screening and isolating of clones. See, forexample: Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor: NY (1989); Maliga et al., Methods in Plant MolecularBiology, Cold Spring Harbor: NY (1995); Birren et al., Genome Analysis:Detecting Genes, Vol. 1, Cold Spring Harbor: NY (1998); Birren et al.,Genome Analysis: Analyzing DNA, Vol. 2, Cold Spring Harbor: NY (1998);Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer: NY(1997).

Examples of oilseed plants include, but are not limited to: soybean,Brassica species, sunflower, maize, cotton, flax and safflower.

Examples of PUFAs having at least twenty carbon atoms and five or morecarbon-carbon double bonds include, but are not limited to, ω-3 fattyacids such as EPA, DPA and DHA. Seeds obtained from such plants are alsowithin the scope of this invention as well as oil obtained from suchseeds.

In one embodiment this invention concerns an oilseed plant comprising:

-   -   a) a first recombinant DNA construct comprising an isolated        polynucleotide encoding a mutant polypeptide having Δ8        desaturase activity, operably linked to at least one regulatory        sequence; and,    -   b) at least one additional recombinant DNA construct comprising        an isolated polynucleotide, operably linked to at least one        regulatory sequence, encoding a polypeptide selected from the        group consisting of: a Δ4 desaturase, a Δ5 desaturase, Δ6        desaturase, a Δ9 desaturase, a Δ12 desaturase, a Δ15 desaturase,        a Δ17 desaturase, a Δ9 elongase, a C_(14/16) elongase, a        C_(16/18) elongase, a C_(18/20) elongase and a C_(20/22)        elongase.

Such additional desaturases are discussed in U.S. Pat. No. 6,075,183,No. 5,968,809, No. 6,136,574, No. 5,972,664, No. 6,051,754, No.6,410,288 and WO 98/46763, WO 98/46764, WO 00/12720 and WO 00/40705.

The choice of combination of cassettes used depends in part on the PUFAprofile and/or desaturase/alongase profile of the oilseed plant cells tobe transformed and the long-chain PUFA(s) which is to be expressed.

In another aspect, this invention concerns a method for makinglong-chain PUFAs in a plant cell comprising:

-   -   (a) transforming a cell with a recombinant construct of the        invention; and,    -   (b) selecting those transformed cells that make long-chain        PUFAs.

In still another aspect, this invention concerns a method for producingat least one PUFA in a soybean cell comprising:

-   -   (a) transforming a soybean cell with a first recombinant DNA        construct comprising:        -   (i) an isolated polynucleotide encoding a mutant polypeptide            having Δ8 desaturase activity, operably linked to at least            one regulatory sequence; and,        -   (ii) at least one additional recombinant DNA construct            comprising an isolated polynucleotide, operably linked to at            least one regulatory sequence, encoding a polypeptide            selected from the group consisting of: a Δ4 desaturase, a Δ5            desaturase, Δ6 desaturase, a Δ9 desaturase, a Δ12            desaturase, a Δ15 desaturase, a Δ17 desaturase, a Δ9            elongase, a C_(14/16) elongase, a C_(16/18) elongase, a            C_(18/20) elongase and a C_(20/22) elongase;    -   (b) regenerating a soybean plant from the transformed cell of        step (a); and,    -   (c) selecting those seeds obtained from the plants of step (b)        having an altered level of PUFAs when compared to the level in        seeds obtained from a nontransformed soybean plant.        In particularly preferred embodiments, the at least one        additional recombinant DNA construct encodes a polypeptide        having Δ9 elongase activity, e.g., the Δ9 elongase isolated        and/or derived from Isochrysis galbana (GenBank Accession No.        AF390174) and set forth in SEQ ID NOs:172-174 or the Δ9 elongase        isolated and/or derived from Euglena gracilis and set forth in        SEQ ID NOs:175-177.        Microbial Expression Systems, Cassettes & Vectors, and        Transformation

The Δ8 desaturase genes and gene products described herein (i.e., thosemutants derived from EgD8 or EgD8S, or homologs thereof) may also beproduced in heterologous microbial host cells, particularly in the cellsof oleaginous yeasts (e.g., Yarrowia lipolytica).

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of any of the geneproducts of the instant sequences. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh-level expression of the encoded enzymes.

Vectors or DNA cassettes useful for the transformation of suitablemicrobial host cells are well known in the art. The specific choice ofsequences present in the construct is dependent upon the desiredexpression products (supra), the nature of the host cell and theproposed means of separating transformed cells versus non-transformedcells. Typically, however, the vector or cassette contains sequencesdirecting transcription and translation of the relevant gene(s), aselectable marker and sequences allowing autonomous replication orchromosomal integration. Suitable vectors comprise a region 5′ of thegene that controls transcriptional initiation (e.g., a promoter) and aregion 3′ of the DNA fragment that controls transcriptional termination(i.e., a terminator). It is most preferred when both control regions arederived from genes from the transformed microbial host cell, although itis to be understood that such control regions need not be derived fromthe genes native to the specific species chosen as a production host.

Initiation control regions or promoters which are useful to driveexpression of the instant Δ8 desaturase ORFs in the desired microbialhost cell are numerous and familiar to those skilled in the art.Virtually any promoter capable of directing expression of these genes inthe selected host cell is suitable for the present invention. Expressionin a microbial host cell can be accomplished in a transient or stablefashion. Transient expression can be accomplished by inducing theactivity of a regulatable promoter operably linked to the gene ofinterest. Stable expression can be achieved by the use of a constitutivepromoter operably linked to the gene of interest. As an example, whenthe host cell is yeast, transcriptional and translational regionsfunctional in yeast cells are provided, particularly from the hostspecies (e.g., see WO 2004/101757 and WO 2006/052870 for preferredtranscriptional initiation regulatory regions for use in Yarrowialipolytica). Any one of a number of regulatory sequences can be used,depending upon whether constitutive or induced transcription is desired,the efficiency of the promoter in expressing the ORF of interest, theease of construction and the like.

Nucleotide sequences surrounding the translational initiation codon‘ATG’ have been found to affect expression in yeast cells. If thedesired polypeptide is poorly expressed in yeast, the nucleotidesequences of exogenous genes can be modified to include an efficientyeast translation initiation sequence to obtain optimal gene expression.For expression in yeast, this can be done by site-directed mutagenesisof an inefficiently expressed gene by fusing it in-frame to anendogenous yeast gene, preferably a highly expressed gene.Alternatively, one can determine the consensus translation initiationsequence in the host and engineer this sequence into heterologous genesfor their optimal expression in the host of interest.

The termination region can be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known and functionsatisfactorily in a variety of hosts (when utilized both in the same anddifferent genera and species from where they were derived). Thetermination region usually is selected more as a matter of conveniencerather than because of any particular property. Preferably, when themicrobial host is a yeast cell, the termination region is derived from ayeast gene (particularly Saccharomyces, Schizosaccharomyces, Candida,Yarrowia or Kluyveromyces). The 3′-regions of mammalian genes encodingγ-interferon and α-2 interferon are also known to function in yeast.Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary; however, it is most preferred if included. Although notintended to be limiting, termination regions useful in the disclosureherein include: ˜100 bp of the 3′ region of the Yarrowia lipolyticaextracellular protease (XPR; GenBank Accession No. M17741); the acyl-coAoxidase (Aco3: GenBank Accession No. AJ001301 and No. CAA04661; Pox3:GenBank Accession No. XP_(—)503244) terminators; the Pex20 (GenBankAccession No. AF054613) terminator; the Pex16 (GenBank Accession No.U75433) terminator; the Lip1 (GenBank Accession No. Z50020) terminator;the Lip2 (GenBank Accession No. AJ012632) terminator; and the3-oxoacyl-coA thiolase (OCT; GenBank Accession No. X69988) terminator.

As one of skill in the art is aware, merely inserting a gene into acloning vector does not ensure that it will be successfully expressed atthe level needed. In response to the need for a high expression rate,many specialized expression vectors have been created by manipulating anumber of different genetic elements that control aspects oftranscription, translation, protein stability, oxygen limitation andsecretion from the microbial host cell. More specifically, some of themolecular features that have been manipulated to control gene expressioninclude: 1.) the nature of the relevant transcriptional promoter andterminator sequences; 2.) the number of copies of the cloned gene andwhether the gene is plasmid-borne or integrated into the genome of thehost cell; 3.) the final cellular location of the synthesized foreignprotein; 4.) the efficiency of translation and correct folding of theprotein in the host organism; 5.) the intrinsic stability of the mRNAand protein of the cloned gene within the host cell; and 6.) the codonusage within the cloned gene, such that its frequency approaches thefrequency of preferred codon usage of the host cell. Each of these typesof modifications are encompassed in the present invention, as means tofurther optimize expression of the mutant Δ8 desaturases describedherein.

Once the DNA encoding a polypeptide suitable for expression in anappropriate microbial host cell (e.g., oleaginous yeast) has beenobtained (e.g., a chimeric gene comprising a promoter, ORF andterminator), it is placed in a plasmid vector capable of autonomousreplication in a host cell, or it is directly integrated into the genomeof the host cell. Integration of expression cassettes can occur randomlywithin the host genome or can be targeted through the use of constructscontaining regions of homology with the host genome sufficient to targetrecombination within the host locus. Where constructs are targeted to anendogenous locus, all or some of the transcriptional and translationalregulatory regions can be provided by the endogenous locus.

In the present invention, the preferred method of expressing genes inYarrowia lipolytica is by integration of linear DNA into the genome ofthe host; and, integration into multiple locations within the genome canbe particularly useful when high level expression of genes are desired[e.g., in the Ura3 locus (GenBank Accession No. AJ306421), the Leu2 genelocus (GenBank Accession No. AF260230), the Lys5 gene (GenBank AccessionNo. M34929), the Aco2 gene locus (GenBank Accession No. AJ001300), thePox3 gene locus (Pox3: GenBank Accession No. XP_(—)503244; or, Aco3:GenBank Accession No. AJ001301), the Δ12 desaturase gene locus(WO2004/104167), the Lip1 gene locus (GenBank Accession No. Z50020)and/or the Lip2 gene locus (GenBank Accession No. AJ012632)].

Advantageously, the Ura3 gene can be used repeatedly in combination with5-fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate;“5-FOA”) selection (infra), to readily permit genetic modifications tobe integrated into the Yarrowia genome in a facile manner.

Where two or more genes are expressed from separate replicating vectors,it is desirable that each vector has a different means of selection andshould lack homology to the other construct(s) to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choice of regulatory regions, selection means and method ofpropagation of the introduced construct(s) can be experimentallydetermined so that all introduced genes are expressed at the necessarylevels to provide for synthesis of the desired products.

Constructs comprising the gene of interest may be introduced into amicrobial host cell by any standard technique. These techniques includetransformation (e.g., lithium acetate transformation [Methods inEnzymology, 194:186-187 (1991)]), protoplast fusion, bolistic impact,electroporation, microinjection, or any other method that introduces thegene of interest into the host cell. More specific teachings applicablefor oleaginous yeasts (i.e., Yarrowia lipolytica) include U.S. Pat. No.4,880,741 and U.S. Pat. No. 5,071,764 and Chen, D. C. et al. (Appl.Microbiol. Biotechnol., 48(2):232-235 (1997)).

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence (e.g., an expression cassette) will be referredto as “transformed” or “recombinant” herein. The transformed host willhave at least one copy of the expression construct and may have two ormore, depending upon whether the gene is integrated into the genome,amplified or is present on an extrachromosomal element having multiplecopy numbers.

The transformed host cell can be identified by various selectiontechniques, as described in WO2004/101757 and WO 2006/052870. Preferredselection methods for use herein are resistance to kanamycin, hygromycinand the amino glycoside G418, as well as ability to grow on medialacking uracil, leucine, lysine, tryptophan or histidine. In alternateembodiments, 5-FOA is used for selection of yeast Ura-mutants. Thecompound is toxic to yeast cells that possess a functioning URΔ3 geneencoding orotidine 5′-monophosphate decarboxylase (OMP decarboxylase);thus, based on this toxicity, 5-FOA is especially useful for theselection and identification of Ura-mutant yeast strains (Bartel, P. L.and Fields, S., Yeast 2-Hybrid System, Oxford University: New York, v.7, pp 109-147, 1997). More specifically, one can first knockout thenative Ura3 gene to produce a strain having a Ura-phenotype, whereinselection occurs based on 5-FOA resistance. Then, a cluster of multiplechimeric genes and a new Ura3 gene can be integrated into a differentlocus of the Yarrowia genome to thereby produce a new strain having aUra+ phenotype. Subsequent integration produces a new Ura3-strain (againidentified using 5-FOA selection), when the introduced Ura3 gene isknocked out. Thus, the Ura3 gene (in combination with 5-FOA selection)can be used as a selection marker in multiple rounds of transformation.

Following transformation, substrates suitable for the instant Δ8desaturases (and, optionally other PUFA enzymes that are co-expressedwithin the host cell) may be produced by the host either naturally ortransgenically, or they may be provided exogenously.

Microbial host cells for expression of the instant genes and nucleicacid fragments may include hosts that grow on a variety of feedstocks,including simple or complex carbohydrates, fatty acids, organic acids,oils and alcohols, and/or hydrocarbons over a wide range of temperatureand pH values. The genes of the present invention have been isolated forexpression in an oleaginous yeast (and in particular Yarrowialipolytica). It is contemplated that because transcription, translationand the protein biosynthetic apparatus is highly conserved, anybacteria, yeast, algae, oomycete and/or fungus will be a suitablemicrobial host for expression of the present nucleic acid fragments.

Preferred microbial hosts are oleaginous yeasts. These organisms arenaturally capable of oil synthesis and accumulation, wherein the oil cancomprise greater than about 25% of the cellular dry weight, morepreferably greater than about 30% of the cellular dry weight, and mostpreferably greater than about 40% of the cellular dry weight. Generatypically identified as oleaginous yeast include, but are not limitedto: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus,Trichosporon and Lipomyces. More specifically, illustrativeoil-synthesizing yeasts include: Rhodosporidium toruloides, Lipomycesstarkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C.tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorulaglutinus, R. graminis, and Yarrowia lipolytica (formerly classified asCandida lipolytica).

Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in afurther embodiment, most preferred are the Y. lipolytica strainsdesignated as ATCC #20352, ATCC #18944, ATCC #76982 and/or LGAM S(7)1(Papanikolaou S., and Aggelis G., Bioresour. Technol. 82(1):43-9(2002)).

Historically, various strains of Y. lipolytica have been used for themanufacture and production of: isocitrate lyase; lipases;polyhydroxyalkanoates; citric acid; erythritol; 2-oxoglutaric acid;γ-decalactone; γ-dodecalatone; and pyruvic acid. Specific teachingsapplicable for engineering ARA, EPA and DHA production in Y. lipolyticaare provided in U.S. patent application Ser. No. 11/264,784 (WO2006/055322), Ser. No. 11/265,761 (WO 2006/052870) and Ser. No.11/264,737 (WO 2006/052871), respectively.

Other preferred microbial hosts include oleaginous bacteria, algae,Oomycetes and other fungi; and, within this broad group of microbialhosts, of particular interest are microorganisms that synthesize ω-3/ω-6fatty acids (or those that can be genetically engineered for thispurpose [e.g., other yeast such as Saccharomyces cerevisiae]). Thus, forexample, transformation of Mortierella alpina (which is commerciallyused for production of ARA) with any of the present Δ8 desaturase genesunder the control of inducible or regulated promoters could yield atransformant organism capable of synthesizing increased quantities ofEDA. The method of transformation of M. alpina is described by Mackenzieet al. (Appl. Environ. Microbiol., 66:4655 (2000)). Similarly, methodsfor transformation of Thraustochytriales microorganisms are disclosed inU.S. Pat. No. 7,001,772.

Based on the teachings described above, in one embodiment this inventionis drawn to a method of producing either DGLA or ETA, respectively,comprising:

-   -   a) providing an oleaginous yeast comprising:        -   (i) a nucleotide sequence encoding a mutant polypeptide            having Δ8 desaturase activity, wherein the mutant            polypeptide has an amino acid sequence as set forth in SEQ            ID NO:2 and wherein SEQ ID NO:2 is not identical to SEQ ID            NO:10; and,        -   (ii) a source of desaturase substrate consisting of either            EDA or ETrA, respectively; and,    -   b) growing the yeast of step (a) in the presence of a suitable        fermentable carbon source wherein the gene encoding the Δ8        desaturase polypeptide is expressed and EDA is converted to DGLA        or ETrA is converted to ETA, respectively; and,    -   c) optionally recovering the DGLA or ETA, respectively, of step        (b).        Substrate feeding may be required.

Of course, since naturally produced PUFAs in oleaginous yeast arelimited to 18:2 fatty acids (i.e., LA), and less commonly, 18:3 fattyacids (i.e., ALA), in more preferred embodiments of the presentinvention the oleaginous yeast will be genetically engineered to expressmultiple enzymes necessary for long-chain PUFA biosynthesis (therebyenabling production of e.g., ARA, EPA, DPA and DHA), in addition to theΔ8 desaturase described herein.

Specifically, in one embodiment this invention concerns an oleaginousyeast comprising:

-   -   a) a first recombinant DNA construct comprising an isolated        polynucleotide encoding a mutant Δ8 desaturase polypeptide,        operably linked to at least one regulatory sequence; and,    -   b) at least one additional recombinant DNA construct comprising        an isolated polynucleotide, operably linked to at least one        regulatory sequence, encoding a polypeptide selected from the        group consisting of: a Δ4 desaturase, a Δ5 desaturase, Δ6        desaturase, a Δ9 desaturase, a Δ12 desaturase, a Δ15 desaturase,        a Δ17 desaturase, a Δ9 elongase, a C_(14/16) elongase, a        C_(16/18) elongase, a C_(18/20) elongase and a C_(20/22)        elongase.        In particularly preferred embodiments, the at least one        additional recombinant DNA construct encodes a polypeptide        having Δ9 elongase activity, e.g., the Δ9 elongase isolated        and/or derived from Isochrysis galbana (GenBank Accession No.        AF390174) and set forth in SEQ ID NOs:172-174 or the Δ9 elongase        isolated and/or derived from Euglena gracilis and set forth in        SEQ ID NOs:175-177.

Metabolic Engineering of Omega-3 and/or Omega-6 Fatty Acid Biosynthesisin Microbes

Methods for manipulating biochemical pathways are well known to thoseskilled in the art; and, it is expected that numerous manipulations willbe possible to maximize ω-3 and/or ω-6 fatty acid biosynthesis inoleaginous yeasts, and particularly, in Yarrowia lipolytica. This mayrequire metabolic engineering directly within the PUFA biosyntheticpathway or additional coordinated manipulation of various othermetabolic pathways.

In the case of manipulations within the PUFA biosynthetic pathway, itmay be desirable to increase the production of LA to enable increasedproduction of ω-6 and/or ω-3 fatty acids. Introducing and/or amplifyinggenes encoding Δ9 and/or Δ12 desaturases may accomplish this. Tomaximize production of ω-6 unsaturated fatty acids, it is well known toone skilled in the art that production is favored in a hostmicroorganism that is substantially free of ALA; thus, preferably, thehost is selected or obtained by removing or inhibiting Δ15 or ω-3 typedesaturase activity that permits conversion of LA to ALA. Alternatively,it may be desirable to maximize production of ω-3 fatty acids (andminimize synthesis of ω-6 fatty acids). In this example, one couldutilize a host microorganism wherein the Δ12 desaturase activity thatpermits conversion of oleic acid to LA is removed or inhibited;subsequently, appropriate expression cassettes would be introduced intothe host, along with appropriate substrates (e.g., ALA) for conversionto ω-3 fatty acid derivatives of ALA (e.g., STA, ETrA, ETA, EPA, DPA,DHA).

In alternate embodiments, biochemical pathways competing with the ω-3and/or ω-6 fatty acid biosynthetic pathways for energy or carbon, ornative PUFA biosynthetic pathway enzymes that interfere with productionof a particular PUFA end-product, may be eliminated by gene disruptionor down-regulated by other means (e.g., antisense mRNA).

Detailed discussion of manipulations within the PUFA biosyntheticpathway as a means to increase ARA, EPA or DHA (and associatedtechniques thereof) are presented in WO 2006/055322, WO 2006/052870 andWO 2006/052871, respectively, as are desirable manipulations in the TAGbiosynthetic pathway and the TAG degradation pathway (and associatedtechniques thereof).

Within the context of the present invention, it may be useful tomodulate the expression of the fatty acid biosynthetic pathway by anyone of the strategies described above. For example, the presentinvention provides methods whereby genes encoding key enzymes in the Δ9elongase/Δ8 desaturase biosynthetic pathway are introduced intooleaginous yeasts for the production of ω-3 and/or ω-6 fatty acids. Itwill be particularly useful to express the present the Δ8 desaturasegenes in oleaginous yeasts that do not naturally possess ω-3 and/or ω-6fatty acid biosynthetic pathways and coordinate the expression of thesegenes, to maximize production of preferred PUFA products using variousmeans for metabolic engineering of the host organism.

Microbial Fermentation Processes for PUFA Production

The transformed microbial host cell is grown under conditions thatoptimize expression of chimeric desaturase and elongase genes andproduce the greatest and the most economical yield of the preferredPUFAs. In general, media conditions that may be optimized include thetype and amount of carbon source, the type and amount of nitrogensource, the carbon-to-nitrogen ratio, the oxygen level, growthtemperature, pH, length of the biomass production phase, length of theoil accumulation phase and the time and method of cell harvest.Microorganisms of interest (i.e., Yarrowia lipolytica) are generallygrown in complex media (e.g., yeast extract-peptone-dextrose broth(YPD)) or a defined minimal media that lacks a component necessary forgrowth and thereby forces selection of the desired expression cassettes(e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).

Fermentation media in the present invention must contain a suitablecarbon source. Suitable carbon sources are taught in WO 2004/101757.Although it is contemplated that the source of carbon utilized in thepresent invention may encompass a wide variety of carbon-containingsources, preferred carbon sources are sugars, glycerol, and/or fattyacids. Most preferred is glucose and/or fatty acids containing between10-22 carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organic(e.g., urea or glutamate) source. In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins and other components knownto those skilled in the art suitable for the growth of the microorganismand promotion of the enzymatic pathways necessary for PUFA production.Particular attention is given to several metal ions (e.g., Mn⁺², Co⁺²,Zn⁺², Mg⁺²) that promote synthesis of lipids and PUFAs (Nakahara, T. etal., Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds. pp 61-97(1992)).

Preferred growth media in the present invention are common commerciallyprepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,Detroit, Mich.). Other defined or synthetic growth media may also beused and the appropriate medium for growth of the transformant hostcells will be known by one skilled in the art of microbiology orfermentation science. A suitable pH range for the fermentation istypically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 ispreferred as the range for the initial growth conditions. Thefermentation may be conducted under aerobic or anaerobic conditions,wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeastcells requires a two-stage process, since the metabolic state must be“balanced” between growth and synthesis/storage of fats. Thus, mostpreferably, a two-stage fermentation process is necessary for theproduction of PUFAs in Yarrowia lipolytica. This approach is describedin WO 2004/101757, as are various suitable fermentation process designs(i.e., batch, fed-batch and continuous) and considerations duringgrowth.

Purification and Processing of PUFA Oils

PUFAs may be found in the host microorganisms and plants as free fattyacids or in esterified forms such as acylglycerols, phospholipids,sulfolipids or glycolipids, and may be extracted from the host cellsthrough a variety of means well-known in the art. One review ofextraction techniques, quality analysis and acceptability standards foryeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology,12(5/6):463-491 (1992)). A brief review of downstream processing is alsoavailable by A. Singh and O. Ward (Adv. Appl. Microbiol., 45:271-312(1997)).

In general, means for the purification of PUFAs may include extractionwith organic solvents, sonication, supercritical fluid extraction (e.g.,using carbon dioxide), saponification and physical means such aspresses, or combinations thereof. One is referred to the teachings of WO2004/101757 for additional details.

Methods of isolating seed oils are well known in the art (Young et al.,Processing of Fats and Oils, In The Lipid Handbook, Gunstone et al.,eds., Chapter 5, pp 253-257; Chapman & Hall: London (1994)). Forexample, soybean oil is produced using a series of steps involving theextraction and purification of an edible oil product from theoil-bearing seed. Soybean oils and soybean byproducts are produced usingthe generalized steps shown in the Table below.

TABLE 5 Generalized Steps For Soybean Oil And Byproduct ProductionImpurities Removed Process And/Or Step Process By-Products Obtained # 1Soybean seed # 2 Oil extraction Meal # 3 Degumming Lecithin # 4 Alkalior physical refining Gums, free fatty acids, pigments # 5 Water washingSoap # 6 Bleaching Color, soap, metal # 7 (Hydrogenation) # 8(Winterization) Stearine # 9 Deodorization Free fatty acids,tocopherols, sterols, volatiles # 10  Oil products

More specifically, soybean seeds are cleaned, tempered, dehulled andflaked, thereby increasing the efficiency of oil extraction. Oilextraction is usually accomplished by solvent (e.g., hexane) extractionbut can also be achieved by a combination of physical pressure and/orsolvent extraction. The resulting oil is called crude oil. The crude oilmay be degummed by hydrating phospholipids and other polar and neutrallipid complexes that facilitate their separation from the nonhydrating,triglyceride fraction (soybean oil). The resulting lecithin gums may befurther processed to make commercially important lecithin products usedin a variety of food and industrial products as emulsification andrelease (i.e., anti-sticking) agents. Degummed oil may be furtherrefined for the removal of impurities (primarily free fatty acids,pigments and residual gums). Refining is accomplished by the addition ofa caustic agent that reacts with free fatty acid to form soap andhydrates phosphatides and proteins in the crude oil. Water is used towash out traces of soap formed during refining. The soapstock byproductmay be used directly in animal feeds or acidulated to recover the freefatty acids. Color is removed through adsorption with a bleaching earththat removes most of the chlorophyll and carotenoid compounds. Therefined oil can be hydrogenated, thereby resulting in fats with variousmelting properties and textures. Winterization (fractionation) may beused to remove stearine from the hydrogenated oil throughcrystallization under carefully controlled cooling conditions.Deodorization (principally via steam distillation under vacuum) is thelast step and is designed to remove compounds which impart odor orflavor to the oil. Other valuable byproducts such as tocopherols andsterols may be removed during the deodorization process. Deodorizeddistillate containing these byproducts may be sold for production ofnatural vitamin E and other high-value pharmaceutical products. Refined,bleached, (hydrogenated, fractionated) and deodorized oils and fats maybe packaged and sold directly or further processed into more specializedproducts. A more detailed reference to soybean seed processing, soybeanoil production and byproduct utilization can be found in Erickson,Practical Handbook of Soybean Processing and Utilization, The AmericanOil Chemists' Society and United Soybean Board (1995). Soybean oil isliquid at room temperature because it is relatively low in saturatedfatty acids when compared with oils such as coconut, palm, palm kerneland cocoa butter.

Plant and microbial oils containing PUFAs that have been refined and/orpurified can be hydrogenated, to thereby result in fats with variousmelting properties and textures. Many processed fats (including spreads,confectionary fats, hard butters, margarines, baking shortenings, etc.)require varying degrees of solidity at room temperature and can only beproduced through alteration of the source oil's physical properties.This is most commonly achieved through catalytic hydrogenation.

Hydrogenation is a chemical reaction in which hydrogen is added to theunsaturated fatty acid double bonds with the aid of a catalyst such asnickel. For example, high oleic soybean oil contains unsaturated oleic,LA and linolenic fatty acids and each of these can be hydrogenated.Hydrogenation has two primary effects. First, the oxidative stability ofthe oil is increased as a result of the reduction of the unsaturatedfatty acid content. Second, the physical properties of the oil arechanged because the fatty acid modifications increase the melting pointresulting in a semi-liquid or solid fat at room temperature.

There are many variables which affect the hydrogenation reaction, which,in turn, alter the composition of the final product. Operatingconditions including pressure, temperature, catalyst type andconcentration, agitation and reactor design are among the more importantparameters that can be controlled. Selective hydrogenation conditionscan be used to hydrogenate the more unsaturated fatty acids inpreference to the less unsaturated ones. Very light or brushhydrogenation is often employed to increase stability of liquid oils.Further hydrogenation converts a liquid oil to a physically solid fat.The degree of hydrogenation depends on the desired performance andmelting characteristics designed for the particular end product. Liquidshortenings (used in the manufacture of baking products, solid fats andshortenings used for commercial frying and roasting operations) and basestocks for margarine manufacture are among the myriad of possible oiland fat products achieved through hydrogenation. A more detaileddescription of hydrogenation and hydrogenated products can be found inPatterson, H. B. W., Hydrogenation of Fats and Oils: Theory andPractice. The American Oil Chemists' Society (1994).

Hydrogenated oils have become somewhat controversial due to the presenceof trans-fatty acid isomers that result from the hydrogenation process.Ingestion of large amounts of trans-isomers has been linked withdetrimental health effects including increased ratios of low density tohigh density lipoproteins in the blood plasma and increased risk ofcoronary heart disease.

PUFA-Containing Oils for Use in Foodstuffs

The market place currently supports a large variety of food and feedproducts, incorporating ω-3 and/or ω-6 fatty acids (particularly ARA,EPA and DHA). It is contemplated that the plant/seed oils, altered seedsand microbial oils of the invention comprising PUFAs will function infood and feed products to impart the health benefits of currentformulations. Compared to other vegetable oils, the oils of theinvention are believed to function similarly to other oils in foodapplications from a physical standpoint (for example, partiallyhydrogenated oils such as soybean oil are widely used as ingredients forsoft spreads, margarine and shortenings for baking and frying).

Plant/seed oils, altered seeds and microbial oils containing ω-3 and/orω-6 fatty acids described herein will be suitable for use in a varietyof food and feed products including, but not limited to: food analogs,meat products, cereal products, snack foods, baked foods and dairyproducts. Additionally, the present plant/seed oils, altered seeds andmicrobial oils may be used in formulations to impart health benefit inmedical foods including medical nutritionals, dietary supplements,infant formula as well as pharmaceutical products. One of skill in theart of food processing and food formulation will understand how theamount and composition of the plant and microbial oil may be added tothe food or feed product. Such an amount will be referred to herein asan “effective” amount and will depend on the food or feed product, thediet that the product is intended to supplement or the medical conditionthat the medical food or medical nutritional is intended to correct ortreat.

Food analogs can be made using processes well known to those skilled inthe art. There can be mentioned meat analogs, cheese analogs, milkanalogs and the like. Meat analogs made from soybeans contain soyprotein or tofu and other ingredients mixed together to simulate variouskinds of meats. These meat alternatives are sold as frozen, canned ordried foods. Usually, they can be used the same way as the foods theyreplace. Meat alternatives made from soybeans are excellent sources ofprotein, iron and B vitamins. Examples of meat analogs include, but arenot limited to: ham analogs, sausage analogs, bacon analogs, and thelike.

Food analogs can be classified as imitation or substitutes depending ontheir functional and compositional characteristics. For example, animitation cheese need only resemble the cheese it is designed toreplace. However, a product can generally be called a substitute cheeseonly if it is nutritionally equivalent to the cheese it is replacing andmeets the minimum compositional requirements for that cheese. Thus,substitute cheese will often have higher protein levels than imitationcheeses and be fortified with vitamins and minerals.

Milk analogs or nondairy food products include, but are not limited to:imitation milk and nondairy frozen desserts (e.g., those made fromsoybeans and/or soy protein products).

Meat products encompass a broad variety of products. In the UnitedStates “meat” includes “red meats” produced from cattle, hogs and sheep.In addition to the red meats there are poultry items which includechickens, turkeys, geese, guineas, ducks and the fish and shellfish.There is a wide assortment of seasoned and processes meat products:fresh, cured and fried, and cured and cooked. Sausages and hot dogs areexamples of processed meat products. Thus, the term “meat products” asused herein includes, but is not limited to, processed meat products.

A cereal food product is a food product derived from the processing of acereal grain. A cereal grain includes any plant from the grass familythat yields an edible grain (seed). The most popular grains are barley,corn, millet, oats, quinoa, rice, rye, sorghum, triticale, wheat andwild rice. Examples of a cereal food product include, but are notlimited to: whole grain, crushed grain, grits, flour, bran, germ,breakfast cereals, extruded foods, pastas, and the like.

A baked goods product comprises any of the cereal food productsmentioned above and has been baked or processed in a manner comparableto baking, i.e., to dry or harden by subjecting to heat. Examples of abaked good product include, but are not limited to: bread, cakes,doughnuts, bars, pastas, bread crumbs, baked snacks, mini-biscuits,mini-crackers, mini-cookies and mini-pretzels. As was mentioned above,oils of the invention can be used as an ingredient.

A snack food product comprises any of the above or below described foodproducts.

A fried food product comprises any of the above or below described foodproducts that has been fried.

A health food product is any food product that imparts a health benefit.Many oilseed-derived food products may be considered as health foods.

A beverage can be in a liquid or in a dry powdered form.

For example, there can be mentioned non-carbonated drinks; fruit juices,fresh, frozen, canned or concentrate; flavored or plain milk drinks,etc. Adult and infant nutritional formulas are well known in the art andcommercially available (e.g., Similac®, Ensure®, Jevity®, and Alimentum®from Ross Products Division, Abbott Laboratories).

Infant formulas are liquids or reconstituted powders fed to infants andyoung children. “Infant formula” is defined herein as an enteralnutritional product which can be substituted for human breast milk infeeding infants and typically is composed of a desired percentage of fatmixed with desired percentages of carbohydrates and proteins in anaquous solution (e.g., see U.S. Pat. No. 4,670,285). Based on theworldwide composition studies, as well as levels specified by expertgroups, average human breast milk typically contains about 0.20% to0.40% of total fatty acids (assuming about 50% of calories from fat);and, generally the ratio of DHA to ARA would range from about 1:1 to 1:2(see, e.g., formulations of Enfamil LIPIL™ [Mead Johnson & Company] andSimilac Advance™ [Ross Products Division, Abbott Laboratories]). Infantformulas have a special role to play in the diets of infants becausethey are often the only source of nutrients for infants; and, althoughbreast-feeding is still the best nourishment for infants, infant formulais a close enough second that babies not only survive but thrive.

A dairy product is a product derived from milk. A milk analog ornondairy product is derived from a source other than milk, for example,soymilk as was discussed above. These products include, but are notlimited to: whole milk, skim milk, fermented milk products such asyogurt or sour milk, cream, butter, condensed milk, dehydrated milk,coffee whitener, coffee creamer, ice cream, cheese, etc.

Additional food products into which the PUFA-containing oils of theinvention could be included are, for example: chewing gums, confectionsand frostings, gelatins and puddings, hard and soft candies, jams andjellies, white granulated sugar, sugar substitutes, sweet sauces,toppings and syrups, and dry-blended powder mixes.

Health Food Products and Pharmaceuticals

A health food product is any food product that imparts a health benefitand includes functional foods, medical foods, medical nutritionals anddietary supplements. Additionally, plant/seed oils, altered seeds andmicrobial oils of the invention may be used in standard pharmaceuticalcompositions. For example, the oils of the invention could readily beincorporated into the any of the above mentioned food products, tothereby produce, e.g., a functional or medical food. More concentratedformulations comprising PUFAs include capsules, powders, tablets,softgels, gelcaps, liquid concentrates and emulsions which can be usedas a dietary supplement in humans or animals other than humans.

Use in Animal Feeds

Animal feeds are generically defined herein as products intended for useas feed or for mixing in feed for animals other than humans. Theplant/seed oils, altered seeds and microbial oils of the invention canbe used as an ingredient in various animal feeds.

More specifically, although not limited herein, it is expected that theoils of the invention can be used within pet food products, ruminant andpoultry food products and aquacultural food products. Pet food productsare those products intended to be fed to a pet [e.g., a dog, cat, bird,reptile, rodent]; these products can include the cereal and health foodproducts above, as well as meat and meat byproducts, soy proteinproducts and grass and hay products (e.g., alfalfa, timothy, oat orbrome grass, vegetables). Ruminant and poultry food products are thosewherein the product is intended to be fed to e.g., turkeys, chickens,cattle and swine. As with the pet foods above, these products caninclude cereal and health food products, soy protein products, meat andmeat byproducts, and grass and hay products as listed above.Aquacultural food products (or “aquafeeds”) are those products intendedto be used in aquafarming, i.e., which concerns the propagation,cultivation or farming of aquatic organisms, animals and/or plants infresh or marine waters.

DESCRIPTION OF PREFERRED EMBODIMENTS

One object of the present invention is the synthesis of suitable Δ8desaturases that will enable expression of the Δ9 elongase/Δ8 desaturasepathway in plants and oleaginous yeast.

In commonly owned WO 2006/012325 and WO 2006/012326 [US2005-0287652]applicants describe the isolation of a Δ8 desaturase from Euglenagracilis (“Eg5”), and the enzyme's functional characterization uponexpression in Saccharomyces cerevisiae. The wildtype Eg5 sequence wasadditionally codon-optimized for expression in Yarrowia lipolytica,resulting in the synthesis of a synthetic, functional codon-optimized Δ8desaturase designated as “D8SF”. Upon co-expression of D8SF with acodon-optimized Δ9 elongase (derived from Isochrysis galbana (GenBankAccession No. 390174) in Yarrowia lipolytica, 6.4% DGLA (with noco-synthesis of GLA) was demonstrated (Example 16 in WO 2006/012325 andWO 2006/012326 [US2005-0287652-A1]).

In the present Application, the synthetic codon-optimized Δ8 desaturasedesignated as “D8SF” (and designated herein as EgD8S”) was subjected totargeted mutations. Ultimately, a mutant EgD8S enzyme (SEQ ID NO:2) wascreated comprising at least one amino acid mutation (and up to about 33amino acid mutations) with respect to the synthetic codon-optimizedEgD8S, wherein: (i) the at least one mutation is selected from the groupconsisting of: 4S to A, 5K to S, 12T to V, 16T to K, 17T to V, 54A to G,55F to Y, 66P to Q, 67S to A, 108S to L, 117G to A, 118Y to F, 120L toM, 121M to L, 125Q to H, 126M to L, 132V to L, 133L to V, 162L to V,163V to L, 170G to A, 171L to V, 279T to L, 280L to T, 293L to M, 3461to V, 3471 to L, 348T to S, 407A to S, 408V to Q, 418A to G, 419G to Aand 422L to Q, wherein the mutations are defined with respect to thesynthetic codon-optimized sequence set forth in SEQ ID NO:10; (ii) SEQID NO:2 is not identical to SEQ ID NO:10; and, (iii) SEQ ID NO:2 is atleast about functionally equivalent to SEQ ID NO:10.

Given the teaching of the present application the skilled person willrecognize the commercial utility of the recombinant genes of the presentinvention encoding Δ8 desaturases, to enable production of a variety ofω-3 and/or ω-6 PUFAs via expression of a heterologous Δ9 elongase/Δ8desaturase pathway.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by: 1.) Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions; Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofmicrobial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds), American Society for Microbiology: Washington,D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, 2^(nd) ed., Sinauer Associates: Sunderland,Mass. (1989). All reagents, restriction enzymes and materials used forthe growth and maintenance of microbial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified.

General molecular cloning was performed according to standard methods(Sambrook et al., supra). Oligonucleotides were synthesized bySigma-Genosys (Spring, Tex.). DNA sequence was generated on an ABIAutomatic sequencer using dye terminator technology (U.S. Pat. No.5,366,860; EP 272,007) using a combination of vector and insert-specificprimers. Sequence editing was performed in Sequencher (Gene CodesCorporation, Ann Arbor, Mich.). All sequences represent coverage atleast two times in both directions. Comparisons of genetic sequenceswere accomplished using DNASTAR software (DNAStar Inc., Madison, Wis.).Alternatively, manipulations of genetic sequences were accomplishedusing the suite of programs available from the Genetics Computer GroupInc. (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.). The GCG program “Pileup” was used with the gap creationdefault value of 12, and the gap extension default value of 4. The GCG“Gap” or “Bestfit” programs were used with the default gap creationpenalty of 50 and the default gap extension penalty of 3. Unlessotherwise stated, in all other cases GCG program default parameters wereused.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “hr” means hour(s), “d” means day(s), “μl” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kB” means kilobase(s). Parts and percentages are by weightand degrees are Celsius.

Transformation and Cultivation of Yarrowia lipolytica

Yarrowia lipolytica strains ATCC #20362, #76982 and #90812 werepurchased from the American Type Culture Collection (Rockville, Md.). Y.lipolytica strains were usually grown at 28° C. on YPD agar (1% yeastextract, 2% bactopeptone, 2% glucose, 2% agar).

Transformation of Yarrowia lipolytica was performed according to themethod of Chen, D. C. et al. (Appl. Microbiol. Biotechnol.,48(2):232-235 (1997)), unless otherwise noted. Briefly, Yarrowia wasstreaked onto a YPD plate and grown at 30° C. for approximately 18 hr.Several large loopfuls of cells were scraped from the plate andresuspended in 1 mL of transformation buffer containing: 2.25 mL of 50%PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2M DTT; and 50 μg sheared salmon sperm DNA. Then, approximately 500 ng oflinearized plasmid DNA was incubated in 100 μl of resuspended cells, andmaintained at 39° C. for 1 hr with vortex mixing at 15 min intervals.The cells were plated onto selection media plates and maintained at 30°C. for 2 to 3 days.

For selection of transformants, minimal medium (“MM”) was generallyused; the composition of MM is as follows: 0.17% yeast nitrogen base(DIFCO Laboratories, Detroit, Mich.) without ammonium sulfate or aminoacids, 2% glucose, 0.1% proline, pH 6.1). Supplements of uracil orleucine were added as appropriate to a final concentration of 0.01%(thereby producing “MMU” or “MMLeu” selection media, respectively, eachprepared with 20 g/L agar).

Alternatively, transformants were selected on 5-fluoroorotic acid(“FOA”; also 5-fluorouracil-6-carboxylic acid monohydrate) selectionmedia, comprising: 0.17% yeast nitrogen base (DIFCO Laboratories,Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose,0.1% proline, 75 mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (ZymoResearch Corp., Orange, Calif.) and 20 g/L agar.

Fatty Acid Analysis of Yarrowia lipolytica

For fatty acid analysis, cells were collected by centrifugation andlipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can.J. Biochem. Physiol., 37:911-917 (1959)). Fatty acid methyl esters wereprepared by transesterification of the lipid extract with sodiummethoxide (Roughan, G., and Nishida I., Arch. Biochem. Biophys.,276(1):3846 (1990)) and subsequently analyzed with a Hewlett-Packard6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard)column. The oven temperature was from 170° C. (25 min hold) to 185° C.at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water, and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) was added to thesample, and then the sample was vortexed and rocked for 20 min. Afteradding 3 drops of 1 M NaCl and 400 μl hexane, the sample was vortexedand spun. The upper layer was removed and analyzed by GC as describedabove.

Example 1 Development of a Topological Model for EgD8S

BLASTP analysis showed that EgD8S contained two domains: an N-terminalcytochrome b₅ domain (located between amino acid residues 5 to 71 of SEQID NO:10) and a C-terminal desaturase domain (located between amino acidresidues 79 to 406 of SEQ ID NO:10). In order to mutate the amino acidsequence of EgD8S without negatively affecting the Δ8 desaturaseactivity, a topological model (FIG. 2) was developed based on the logicand analyses below.

First, the TMHMM program (“Prediction of transmembrane helices inproteins”; TMHMM Server v. 2.0, Center for Biological Sequence Analysis,BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby,Denmark) predicted that EgD8S had four membrane-spanning helices (aminoacid residues 113-132, 223-245, 266-283 and 287-309), with both the N-and C-termini located on the cytoplasmic side of the membrane.

The membrane-bound fatty acid desaturases belong to a super-family ofmembrane di-iron proteins that feature three His-rich motifs: HX₍₃₋₄₎H(SEQ ID NOs:166 and 167), HX₍₂₋₃₎HH (SEQ ID NOs:168 and 169) and(H/Q)X₍₂₋₃₎HH (SEQ ID NOs:170 and 171). These His-rich residues havebeen predicted to be located in the cytoplasmic face of the membrane andhave been shown to be important for enzyme activity (Shanklin, J. etal., Biochemistry, 33:12787-12794 (1994); Shanklin, J., and Cahoon, E.B., Annu. Rev. Plant Physiol. Plant Mol. Biol., 49:611-641 (1998)).Within SEQ ID NO:10, these three histidine boxes were located betweenamino acid residues 146-150, 183-187 and 358-362; two additional Hisresidues are located at amino acid residues 27 and 50. Each of these Hisresidues are depicted on FIG. 2 with a small round circle.

If the model predicted by TMHMM (supra) were accepted withoutalteration, the first two His-rich regions (i.e., the regions spanningbetween amino acid residues 146-150 and 183-187) would be located in theperiplasmic space, thus preventing their participation in theiron-active site.

The conflict noted above was resolved when hydropathy plot analysis(Kyte, J., and Doolittle, R., J. Mol. Biol., 157:105-132 (1982))predicted one more hydrophobic region located between amino acidresidues 88-109 that immediately preceded the first predictedtransmembrane segment (i.e., residues 113-132). Since the N-terminalcytochrome-b₅ domain is located in the cytoplasmic space, it waspredicted that the hydrophobic region (i.e., residues 88-109) should bethe first membrane-spanning segment (i.e., region I, as shown in FIG.2), while the predicted transmembrane segment corresponding to residues113-132 was designated as the second membrane-spanning segment (i.e.,region II, as shown in FIG. 2). As a result, the transmembrane segmentfound between residues 223-245 that was originally predicted by TMHMM tospan through the membrane was instead predicted to lie in thecytoplasmic face, such that the first two His-rich motifs (i.e., theregions spanning between amino acid residues 146-150 and 183-187) couldbe adjusted to be within the cytoplasmic side.

Finally, the hydropathy plot analysis also predicted another hydrophobicregion (i.e., residues 157-172) between the first two His-rich motifs.Because the substrate for the desaturase is highly hydrophobic, it wasexpected to most likely partition into the lipid bilayer of thecytoplasmic membrane. This suggested that the desaturase active siteassembled from the His-rich motifs might be at (or very near) themembrane surface. Thus, it was hypothesized that both hydrophobicregions (i.e., residues 157-172 and residues 223-245) lie near themembrane surface to ensure that the active site sits close the membrane.

The last two membrane-spanning helices predicted by TMHMM (i.e.,residues 266-283 and 287-309) are included within the final topologicalmodel shown in FIG. 2 as region III and region IV.

Thus, the topological model depicted in FIG. 2 includes fourtransmembrane regions labeled as regions I, II, III and IV, whichcorrespond to amino acid residues 88-109, 113-132, 266-283 and 287-309,respectively. Two additional hydrophobic regions are located at aminoacid residues 157-172 and 223-245. Finally, “IN” corresponds with thecytoplasmic space, while “OUT” corresponds with the periplasmic space.

Example 2 Strategies to Select Amino Acid Residues for Mutation

Close homologs to the EgD8S sequence were determined by conducting BLAST(Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol.Biol., 215:403-410 (1993)) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the SWISS-PROT protein sequencedatabase, EMBL and DDBJ databases). Specifically, EgD8S (SEQ ID NO:10)was compared for similarity to all publicly available protein sequencescontained in the “nr” database, using the BLASTP algorithm (Altschul etal., J. Mol. Biol. 215:403-410 (1990)) provided by the NCBI.

Ignoring all hits to any Δ8 desaturase isolated from Euglena gracilis,the BLASTP searches showed that EgD8S was most homologous to thefollowing proteins:

TABLE 6 Homologous Proteins To EgD8S, Based On BLASTP Analysis % GenBank% Simi- Accession No. Protein Organism Identity larity E-Value CAE65324hypothetical Caenorhabditis 38 56 1E−65 protein briggsae CBG10258AAR27297 Δ6 Amylomyces 35 52 3E−65 desaturase rouxii AAS93682 Δ6Rhizopus 32 53 4E−64 desaturase orizae * “% Identity” is defined as thepercentage of amino acids that are identical between the two proteins.** “% Similarity” is defined as the percentage of amino acids that areidentical or conserved between the two proteins. *** “Expectation value”estimates the statistical significance of the match, specifying thenumber of matches, with a given score, that are expected in a search ofa database of this size absolutely by chance.

In order to select the amino acid residues that could be mutated withinEgD8S without affecting the Δ8 desaturase activity, a set of criteriawere developed to identify preferred targets for mutation, as outlinedbelow.

1. Preferred amino acid residue targets of the EgD8S desaturase domain(located between amino acid residues 79 to 406 of SEQ ID NO:10) are notconserved, when compared to the Δ6 desaturase of A. rouxii (supra;“ArD6”; SEQ ID NO:13), the Δ6 desaturase of R. orizae (supra; “RoD6”;SEQ ID NO:14) and representatives of other desaturases such as the Δ8fatty acid desaturase-like protein of Leishmania major (GenBankAccession No. CAJ09677; “LmD8L”; SEQ ID NO:15), and the Δ6 desaturase ofMortierella isabellina (GenBank Accession No. AAG38104; “MiD6”; SEQ IDNO:16). An alignment of these proteins is shown in FIG. 3, using themethod of Clustal W (slow, accurate, Gonnet option; Thompson et al.,Nucleic Acids Res., 22:4673-4680 (1994)) of the MegAlign™ program ofDNASTAR™ software. It was hypothesized that changes in the non-conservedregions among these 5 different desaturases should not affect the Δ8desaturase activity of EgD8S.

2. Preferred amino acid residue targets of the cytochrome b₅ domain ofEgD8S (located between amino acids 5 to 71 of SEQ ID NO:10) are notconserved, when compared to the cytochrome b₅ genes of Saccharomycescerevisiae (GenBank Accession No. P40312; “SCb5”; SEQ ID NO:178) andSchizosaccharomyces pombe (GenBank Accession No. 094391; SPb5; SEQ IDNO:179). An alignment of the N-terminal portion of EgD8S (i.e., aminoacid residues 1-136 of SEQ ID NO:10) with SCb5 and SPb5 is shown in FIG.4, using the method of Clustal W (supra) of the MegAlign™ program ofDNASTAR™ software. It was hypothesized that changes in the non-conservedregion among these 3 different proteins should not affect the electrontransport function of the cytochrome b₅ domain of EgD8S and thus notaffect the Δ8 desaturase activity.

3. Preferred amino acid residue targets are on the transmembrane helicesclose to the endoplasmic reticulum (ER) side of the membrane or exposedto the ER lumen.

4. Preferred amino acid residue targets are close to the N-terminal orC-terminal ends of the EgD8S enzyme, since non-conserved residues inthese regions may tolerate more mutations.

Based on the above criteria, a set of 70 target mutation sites(comprising one, two or three amino acid residues) within EgD8S (i.e.,SEQ ID NO:10) were selected for mutation as described below in Table 7.Of the individual 126 amino acid residue mutations, 53 (i.e., 42.1%)were identified as “non-conservative amino acid substitutions” while 73(i.e., 57.9%) were identified as “conservative amino acidsubstitutions”.

TABLE 7 Selected Amino Acid Residues Suitable For Targeted MutationMutation Sequence Mutations Site Within SEQ ID NO: 10 M1 4S to A, 5K toS M2 12T to V M3 16T to K, 17T to V M4 25N to D, 26F to E M5 31A to D,32E to S M6 59K to L M7 61M to A, 62P to V M8 66P to Q, 67S to A M9 72Pto Q, 73Q to P M10 79A to Q, 80Q to A M11 87R to A, 88E to I M12 407A toS, 408V to Q M13 412M to S, 413A to Q M14 416Q to V, 417P to Y M15 422Lto Q M16 108S to L M17 110T to A M18 120L to M, 121M to L M19 122V to SM20 123Q to Y, 124Y to Q M21 125Q to H, 126M to L M22 127Y to Q M23 288Sto N M24 289I to P, 290L to M M25 291T to V, 292S to V M26 293L to M M27296F to T M28 298V to S M29 392N to T, 393P to T M30 394L to G, 395P toM M31 7Q to L, 8A to S M32 10P to W, 11L to Q M33 21S to F, 22A to S M3446F to S, 47M to L M35 48V to F, 49M to L M36 37Y to F, 38Q to N M37 51Sto T, 52Q to N M38 54A to G, 55F to Y M39 64I to L, 65N to D M40 69E toD, 70L to V M41 75A to G, 76V to L M42 89E to D, 90L to I M43 97D to E,98A to V M44 110T to S, 111L to V M45 117G to A, 118Y to F M46 132V toL, 133L to V M47 198D to E, 199I to L M48 231L to V, 232V to L M49 297Fto V, 298V to L M50 309I to V, 310V to I M51A 347I to L, 348T to S M51B346I to V, 347I to L, 348T to S M52 400V to I, 401I to V M53 9L to V M5419D to E, 20V to I M55 33I to L M56 45A to G, 46F to Y M57 57K to R, 58Lto I M58 65N to Q M59 73Q to N, 74A to G M60 96F to Y M61 239F to I,240I to F M62 271L to M, 272A to S M63 279T to L, 280L to T M64 130G toA, 131A to G M65 304G to F, 305F to G M66 229F to Y, 230Y to F M67 291Tto S, 292S to L M68 162L to V, 163V to L M69 170G to A, 171L to V M70418A to G, 419G to A

Example 3 Generation of Yarrowia lipolytica Strains Y4001 and Y4001U toProduce about 17% EDA of Total Lipids

The present Example describes the construction of strains Y4001 andY4001U, derived from Yarrowia lipolytica ATCC #20362, and each capableof producing 17% EDA (C20:2) relative to the total lipids. Both strainswere engineered to test functional expression of EgD8S and mutationsthereof. Thus, it was necessary to construct host strains capable ofproducing the Δ8 desaturase substrate, EDA.

The development of strain Y4001U, having a Leu- and Ura-phenotype,required the construction of strain Y2224 (a FOA resistant mutant froman autonomous mutation of the Ura3 gene of wildtype Yarrowia strain ATCC#20362) and strain Y4001.

Generation of Strain Y2224

Strain Y2224 was isolated in the following manner: Yarrowia lipolyticaATCC #20362 cells from a YPD agar plate (1% yeast extract, 2%bactopeptone, 2% glucose, 2% agar) were streaked onto a MM plate (75mg/L each of uracil and uridine, 6.7 g/L YNB with ammonia sulfate,without amino acid, and 20 g/L glucose) containing 250 mg/L 5-FOA (ZymoResearch). Plates were incubated at 28° C. and four of the resultingcolonies were patched separately onto MM plates containing 200 mg/mL5-FOA and MM plates lacking uracil and uridine to confirm uracil Ura3auxotrophy.

Generation of Strain Y4001 to Produce 17% EDA of Total Lipids

Strain Y4001 was created via integration of construct pZKLeuN-29E3 (FIG.5A; comprising four chimeric genes [a Δ12 desaturase, a C_(16/18)elongase and two Δ9 elongases]) into the Leu2 loci of Y2224 strain tothereby enable production of EDA.

Construct pZKLeuN-29E3 contained the following components:

TABLE 8 Description of Plasmid pZKLeuN-29E3 (SEQ ID NO: 17) RE Sites AndNucleotides Within SEQ ID Description Of NO: 17 Fragment And ChimericGene Components BsiW I/Asc I 795 bp 3′ part of Yarrowia Leu2 gene(GenBank 7797-7002 Accession No. AF260230) Sph I/Pac I 703 bp 5′ part ofYarrowia Leu2 gene (GenBank 4302-3591 Accession No. AF260230) Swa I/BsiWI GPD::F.D12::Pex20, comprising: (10500-7797) GPD: Yarrowia lipolyticaGPD promoter (WO 2005/003310) F.D12: Fusarium moniliforme Δ12 desaturasegene (WO 2005/047485) Pex20: Pex20 terminator sequence from YarrowiaPex20 gene (GenBank Accession No. AF054613) Bgl II/Swa I Exppro::EgD9E::Lip1, comprising: (12526-10500) Exp pro: Yarrowia lipolyticaexport protein (EXP1) promoter (WO 2006/052870 and U.S. Pat. applicationSer. No. 11/265,761) EgD9E: codon-optimized Δ9 elongase gene (SEQ ID NO:177), derived from Euglena gracilis (SEQ ID NOs: 175 and 176; U.S. Pat.application Ser. No. 60/739,989; see also Example 16 herein) Lip1: Lip1terminator sequence from Yarrowia Lip1 gene (GenBank Accession No.Z50020) Pme I/Cla I FBAINm::EgD9S::Lip2, comprising: (12544-1) FBAINm:Yarrowia lipolytica FBAINm promoter (WO 2005/049805) EgD9S:codon-optimized Δ9 elongase gene (SEQ ID NO: 177; supra) Lip2: Lip2terminator sequence from Yarrowia Lip2 gene (GenBank Accession No.AJ012632) Cla I/EcoR I LoxP::Ura3::LoxP, comprising: (1-1736) LoxPsequence (SEQ ID NO: 18) Yarrowia Ura3 gene (Gen Bank Accession No.AJ306421) LoxP sequence (SEQ ID NO: 18) EcoR I/Pac I NT::ME3S::Pex16,comprising: (1736-3591) NT: Yarrowia lipolytica YAT1 promoter (PatentPublication U.S. 2006/0094102-A1) ME3S: codon-optimized C_(16/18)elongase gene (SEQ ID NO: 19), derived from M. alpina (see U.S. Pat.application Ser. No. 11/253,882 and also WO 2006/052870) Pex16: Pex16terminator sequence of Yarrowia Pex 16 gene GenBank Accession No. U75433

Plasmid pZKLeuN-29E3 was digested with Asc I/Sph I, and then used fortransformation of Y. lipolytica strain Y2224 (i.e., ATCC #20362 Ura3-)according to the General Methods. The transformant cells were platedonto MMLeu media plates and maintained at 30° C. for 2 to 3 days. Thecolonies were picked and streaked onto MM and MMLeu selection plates.The colonies that could grow on MMLeu plates but not on MM plates wereselected as Leu-strains. Single colonies of Leu-strains were theninoculated into liquid MMLeu at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, lipids were extracted,and fatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed the presence of EDA in the transformants containingthe 4 chimeric genes of pZKLeuN-29E3, but not in the Yarrowia Y2224control strain. Most of the selected 36 Leu-strains produced about 12 to16.9% EDA of total lipids. There were 3 strains (i.e., strains #11, #30and #34) that produced about 17.4%, 17% and 17.5% EDA of total lipids;they were designated as strains Y4001, Y4002 and Y4003, respectively.

Generation of Strain Y4001U (Leu-, Ura-) to Produce 17% EDA of TotalLipids

Strain Y4001U was created via temporary expression of the Crerecombinase enzyme in plasmid pY116 (FIG. 5B) within strain Y4001 toproduce a Leu- and Ura-phenotype. Construct pY116 contained thefollowing components:

TABLE 9 Description of Plasmid pY116 (SEQ ID NO: 180) RE Sites AndNucleotides Within SEQ ID Description Of Fragment NO: 180 And ChimericGene Components 1328-448 ColE1 plasmid origin of replication 2258-1398Ampicillin-resistance gene (Amp^(R)) 3157-4461 Yarrowia autonomousreplication sequence (ARS18; GenBank Accession No. A17608) PacI/SawIYarrowia Leu2 gene (GenBank Accession No. 6667-4504 AF260230) 6667-180GPAT::Cre::XPR2, comprising: GPAT: Yarrowia lipolytica GPAT promoter (WO2006/031937) Cre: Enterobacteria phage P1 Cre gene for recombinaseprotein (GenBank Accession No. X03453) XPR2: ~100 bp of the 3′ region ofthe Yarrowia Xpr gene (GenBank Accession No. M17741)

Plasmid pY116 was used for transformation of freshly grown Y4001 cellsaccording to the General Methods. The transformant cells were platedonto MMU plates containing 280 μg/mL sulfonylurea and maintained at 30°C. for 3 to 4 days. Four colonies were picked, inoculated into 3 mLliquid YPD media at 30° C. and shaken at 250 rpm/min for 1 day. Thecultures were diluted to 1:50,000 with liquid MMU media, and 100 μL wasplated onto new YPD plates and maintained at 30° C. for 2 days. Colonieswere picked and streaked onto MMLeu and MMLeu+Ura selection plates. Thecolonies that could grow on MMLeu+Ura plates but not on MMLeu plateswere selected and analyzed by GC to confirm the presence of C20:2 (EDA).One strain, having a Leu- and Ura-phenotype, produced about 17% EDA oftotal lipids and was designated as Y4001U.

Example 4 Generation of Auto-Replicating Plasmid pFmD8S

The present Example describes the construction of plasmid pFmD8Scomprising a chimeric FBAINm::EgD8S::XPR gene. Plasmid pFmD8S (SEQ IDNO:20; FIG. 6D) was constructed by three-way ligation using fragmentsfrom plasmids pKUNFmkF2, pDMW287F and pDMW214. Plasmid pFmD8S, anauto-replicating plasmid that will reside in Yarrowia in 1-3 copies, wasutilized to test functional expression of EgD8S (and mutations thereof),as described in Examples 5-10, infra.

Plasmid pKUNFmkF2

pKUNFmkF2 (SEQ ID NO:21; FIG. 6A; WO 2006/012326) is a constructcomprising a chimeric FGAINm::Lip2 gene (wherein “FBAINmK” is theYarrowia lipolytica FBAINm promoter [WO 2005/049805], “F.D12” is theFusarium moniliforme Δ12 desaturase [WO 2005/047485], and “Lip2” is theYarrowia lipolytica Lip2 terminator sequence (GenBank Accession No.AJ012632)).

Plasmid pDMW287F

pDMW287F (SEQ ID NO:22; FIG. 6B; WO 2006/012326) is a constructcomprising the synthetic Δ8 desaturase, derived from wildtype Euglenagracilis, and codon-optimized for expression in Yarrowia lipolytica(wherein EgD8S is identified as “D8SF” in the Figure). The desaturasegene is flanked by a Yarrowia lipolytica FBAIN promoter (WO 2005/049805;identified as “FBA1+intron” in the Figure) and a Pex16 terminatorsequence of the Yarrowia Pex16 gene (GenBank Accession No. U75433).

Plasmid pDMW214

pDMW214 (SEQ ID NO:23; FIG. 6C; WO 2005/049805) is a shuttle plasmidthat could replicate both in E. coli and Yarrowia lipolytica. Itcontained the following components:

TABLE 10 Description Of Plasmid pDMW214 (SEQ ID NO: 23) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And NO: 23 ChimericGene Components 1150-270 ColE1 plasmid origin of replication 2080-1220Ampicillin-resistance gene (Amp^(R)) 2979-4256 Yarrowia autonomousreplication sequence (ARS18; GenBank Accession No. A17608) PmeI/SphIYarrowia Leu2 gene (GenBank Accession No. 6501-4256 AF260230) 6501-1FBA1 + intron::GUS::XPR2, comprising: FBA1 + intron: Yarrowia lipolyticaFBAIN promoter (WO 2005/049805) GUS: E. coli gene encodingβ-glucuronidase (Jefferson, R. A. Nature. 342: 837-838 (1989) XPR2: ~100bp of the 3′ region of the Yarrowia Xpr gene (GenBank Accession No.M17741)

Final Construction of Plasmid pFmD8S

The PmeI/NcoI fragment of plasmid pKUNFmkF2 (FIG. 6A; comprising theFBAINm promoter) and the NcoI/NotI fragment of plasmid pDMW287F (FIG.6B; comprising the synthetic Δ8 desaturase gene EgD8S) were useddirectionally to replace the PmeI/Not I fragment of pDMW214 (FIG. 6C).This resulted in generation of pFmD8S (SEQ ID NO:20; FIG. 6D),comprising a chimeric FBAINm::EgD8S::XPR2 gene. Thus, the components ofpFmD8S are as described in Table 11 below.

TABLE 11 Components Of Plasmid pFmD8S (SEQ ID NO: 20) RE Sites AndNucleotides Within SEQ ID Description Of NO: 20 Fragment And ChimericGene Components Swa I/Sac II FBAINm::EgD8S::XPR2, comprising:(7988-1461) FBAINm: Yarrowia lipolytica FBAINm promoter (WO 2005/049805)EgD8S: codon-optimized Δ8 desaturase gene (SEQ ID NO: 9, identified as“D8-corrected” in FIG. 6D), derived from E. gracilis (SEQ ID NO: 11)XPR2: ~100 bp of the 3′ region of the Yarrowia Xpr gene (GenBankAccession No. M17741) 2601-1721 ColE1 plasmid origin of replication3531-2671 Ampicillin-resistance gene (Amp^(R)) for selection in E. coli4430-5734 Yarrowia autonomous replication sequence (ARS18; GenBankAccession No. A17608) 7942-5741 Yarrowia Leu2 gene (GenBank AccessionNo. AF260230)

Example 5 Development of a Quick Screen to Functionally Analyze Δ8Desaturase Activity in Yarrowia lipolytica

A set of 40 mutations was created using pFmD8S (Example 4) as templateand 40 pairs of oligonucleotide primers to individually mutate targetedamino acid residues within EgD8S (SEQ ID NO:10) by site-directedmutagenesis (QuikChange® Kit, Stratagene, La Jolla, Calif.). Specificmutations were selected from those set forth in Table 7 of Example 2 andprimer pairs were selected from the oligonucleotides set forth in SEQ IDNOs:24-164, such that creation of the M1 mutation (i.e., 4S to A, 5K toS within SEQ ID NO:10) required primers 1A and 1B (SEQ ID NOs:24 and 25,respectively), etc. Plasmids from each mutation were transformed into E.coli XL2Blue cells (Stratagene). Four colonies from each of the 40transformations were picked and grown individually in liquid media at37° C. overnight. Plasmids (i.e., 160 total) were isolated from thesecultures and sequenced individually to confirm the mutations.

Plasmid pFmD8S and the isolated mutant plasmids were transformed intostrain Y4001 (Example 3) individually, as described in the GeneralMethods. The transformants were selected on MM plates. After 2 daysgrowth at 30° C., transformants were scraped from each plate, lipidswere extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that there were about 7% DGLA and 12% EDA of totallipids produced by the transformants with plasmid pFmD8S; the averageconversion efficiency whereby EgD8S converted EDA to DGLA in thetransformants was 36.8%. The conversion efficiency was measuredaccording to the following formula: ([product]/[substrate+product])*100,where ‘product’ includes the immediate product and all products in thepathway derived from it.

GC analyses of transformants carrying mutations within EgD8S showed that30 of the 40 mutations did not affect the Δ8 desaturase activity (whencompared to the synthetic codon-optimized EgD8S Δ8 desaturase activityin transformants carrying plasmid pFmD8S). These results suggested thatthe screening procedure described herein (i.e., with pFmD8S as parentplasmid and strain Y4001 as a host) could be used to quickly screen theEgD8S mutants and identify which mutations negatively affected Δ8desaturase activity.

Based on these results, the remaining 30 mutations set forth in Table 7of Example 2 were synthesized (although some mutations were introducedin combination for efficiency), using the methodology described above.Table 12 describes the Δ8 desaturase activity attributed to eachmutation site (i.e., M1 to M70), as a percent of the Δ8 desaturaseactivity resulting in each mutant EgD8S with respect to the Δ8desaturase activity of the synthetic codon-optimized EgD8S (SEQ IDNO:10). As seen in the Table below, Δ8 desaturase activity ranged from0% up to 125%.

TABLE 12 Mutant Δ8 Desaturase Activities Mutations Primers Used %Activity M1 1A, 1B 115% (SEQ ID NOs: 24 and 25) M2 2A, 2B 110% (SEQ IDNOs: 26 and 27) M3 3A, 3B 100% (SEQ ID NOs: 28 and 29) M4 4A, 4B N/A(SEQ ID NOs: 30 and 31) M5 5A, 5B N/A (SEQ ID NOs: 32 and 33) M6 6A, 6B110% (SEQ ID NOs: 34 and 35) M7 7A, 7B  30% (SEQ ID NOs: 36 and 37) M88A, 8B 100% (SEQ ID NOs: 38 and 39) M9 9A, 9B N/A (SEQ ID NOs: 40 and41) M10 10A, 10B N/A (SEQ ID NOs: 42 and 43) M11 11A, 11B 20% (SEQ IDNOs: 44 and 45) M12 12A, 12B 100% (SEQ ID NOs: 46 and 47) M13 13A, 13BN/A (SEQ ID NOs: 48 and 49) M14 14A, 14B 100% (SEQ ID NOs: 50 and 51)M15 15A, 15B 125% (SEQ ID NOs: 52 and 53) M16 16A, 16B 100% (SEQ ID NOs:54 and 55) M17 17A, 17B  50% (SEQ ID NOs: 56 and 57) M18 18A, 18B N/A(SEQ ID NOs: 58 and 59) M19 19A, 19B 100% (SEQ ID NOs: 60 and 61) M2020A, 20B 80% (SEQ ID NOs: 62 and 63) M21 21A, 21B 120% (SEQ ID NOs: 64and 65) M22 22A, 22B 110% (SEQ ID NOs: 66 and 67) M23 23A, 23B  80% (SEQID NOs: 68 and 69) M24 24A, 24B N/A (SEQ ID NOs: 70 and 71) M25 25A, 25BN/A (SEQ ID NOs: 72 and 73) M26 26A, 26B 110% (SEQ ID NOs: 74 and 75)M27 27A, 27B  80% (SEQ ID NOs: 76 and 77) M28 28A, 28B  90% (SEQ ID NOs:78 and 79) M29 29A, 29B N/A (SEQ ID NOs: 80 and 81) M30 30A, 30B  85%(SEQ ID NOs: 82 and 83) M31 31A, 31B  85% (SEQ ID NOs: 84 and 85) M3232A, 32B N/A (SEQ ID NOs: 86 and 87) M33 33A, 33B N/A (SEQ ID NOs: 88and 89) M34 34A, 34B N/A (SEQ ID NOs: 90 and 91) M35 35A, 35B  0% (SEQID NOs: 92 and 93) M36 36A, 36B  80% (SEQ ID NOs: 94 and 95) M37 37A,37B  90% (SEQ ID NOs: 96 and 97) M38 38A, 38B 100% (SEQ ID NOs: 98 and99) M39 39A, 39B 100% (SEQ ID NOs: 100 and 101) M40 40A, 40B 100% (SEQID NOs: 102 and 103) M41 41A, 41B 100% (SEQ ID NOs: 104 and 105) M4242A, 42B  0% (SEQ ID NOs: 106 and 107) M43 43A, 43B  90% (SEQ ID NOs:108 and 109) M44 44A, 44B N/A (SEQ ID NOs: 110 and 111) M45 45A, 45B100% (SEQ ID NOs: 112 and 113) M46 46A, 46B 100% (SEQ ID NOs: 114 and115) M47 47A, 47B  0% (SEQ ID NOs: 116 and 117) M48 48A, 48B N/A (SEQ IDNOs: 118 and 119) M49 49A, 49B 100% (SEQ ID NOs: 120 and 121) M50 50A,50B 100% (SEQ ID NOs: 122 and 123) M51 51A, 51B 100% (SEQ ID NOs: 124and 125) M51B 51A, 51B 100% (SEQ ID NOs: 126 and 125) M52 52A, 52B  80%(SEQ ID NOs: 127 and 128) M53 53A, 53B 100% (SEQ ID NOs: 129 and 130)M54 54A, 54B 100% (SEQ ID NOs: 131 and 132) M55 55A, 55B  40% (SEQ IDNOs: 133 and 134) M56 56A, 56B  0% (SEQ ID NOs: 135 and 136) M57 57A,57B N/A (SEQ ID NOs: 137 and 138) M58 58A, 58B 100% (SEQ ID NOs: 139 and140) M59 59A, 59B  90% (SEQ ID NOs: 141 and 142) M60 60A, 60B  50% (SEQID NOs: 143 and 144) M61 61A, 61B  50% (SEQ ID NOs: 145 and 146) M6262A, 62B  50% (SEQ ID NOs: 147 and 148) M63 63A, 63B 100% (SEQ ID NOs:149 and 150) M64 64A, 64B  60% (SEQ ID NOs: 151 and 152) M65 65A, 65B 0% (SEQ ID NOs: 153 and 154) M66 66A, 66B N/A (SEQ ID NOs: 155 and 156)M67 67A, 67B N/A (SEQ ID NOs: 157 and 158) M68 68A, 68B 100% (SEQ IDNOs: 159 and 160) M69 69A, 69B 100% (SEQ ID NOs: 161 and 162) M70 70A,70B 100% (SEQ ID NOs: 163 and 164) * N/A is reported when the desiredmutation was not successfully produced or when GC data was lacking.

Example 6 Generation of pFmD8S-1, pFmD8S-001, pFmD8S-2A, pFmD8S-2B,pFmD8S-3A, pFmD8S-3B, pFmD8S-003, pFmD8S-4, pFmD8S-004, pFmD8S-005 andpFmD8S-006 Constructs by Site-Directed Mutagenesis of EgD8S withinConstruct pFmD8S

A series of plasmids were generated by consecutive rounds of continuedsite-directed mutagenesis to introduce multiple select mutations intoEgD8S (SEQ ID NOs:9 and 10). pFmD8S (Example 4), comprising thesynthetic codon-optimized EgD8S, was used as the starting template inTables 13, 14, 16 and 17, while a mutant created thereof (i.e.,pFmD8S-M45, comprising 117G to A and 118Y to F mutations with respect toSEQ ID NO:10) was used as the starting template in Table 15. Theresulting plasmids comprising mutant EgD8S sequences, as well as detailsconcerning the primers used to produce these mutations, are describedbelow in Tables 13, 14, 15, 16 and 17.

The column titled “Mutation Site Introduced” refers to the specificamino acid sites selected for mutation, as listed in Table 7 of Example2. In the column titled “Total Mutations In Resultant Plasmid WithRespect to EgD8S (SEQ ID NO:10)”, those amino acid mutations that arehighlighted in bold-face text correspond to newly introduced mutationsthat were not present in the template in the indicated round ofsite-directed mutagenesis. The number shown in parentheses correspondswith the number of total mutations in the resultant plasmid with respectto EgD8S (SEQ ID NO:10).

TABLE 13 Generation Of pFmD8S-1 And pFmD8S-001 Constructs Mutation SiteResultant Total Mutations In Resultant Plasmid Round Introduced TemplatePrimers Plasmid With Respect to EgD8S (SEQ ID NO: 10) 1 M3 pFmD8S 3A, 3BpFmD8S-M3 16 T to K, 17T to V (2) (SEQ ID NOs: 28 and 29) 2 M1 pFmD8S-M31A, 1B pFmD8S-M3, 1 16 T to K, 17T to V, 4S to A, 5K to S (4) (SEQ IDNOs: 24 and 25) 3 M2 pFmD8S-M3, 1 2A, 2B pFmD8S-M3, 1, 2 16 T to K, 17Tto V, 4S to A, 5K to S, (SEQ ID NOs: 26 and 27) 12T to V (5) 4 M8pFmD8S- 8A, 8B pFmD8S-1 16 T to K, 17T to V, 4S to A, 5K to S, M3, 1, 2(SEQ ID NOs: 38 and 39) 12T to V, 66P to Q, 67S to A (7) 5 M38 pFmD8S-138A, 38B pFmD8S-001 16 T to K, 17T to V, 4S to A, 5K to S, (SEQ ID NOs:98 and 99) 12T to V, 65P to Q, 66S to A, 54A to G, 55F to Y (9)

TABLE 14 Generation Of pFmD8S-2A And pFmD8S-003 Constructs Mutation SiteResultant Total Mutations In Resultant Plasmid Round Introduced TemplatePrimers Plasmid With Respect to EgD8S (SEQ ID NO: 10) 1 M45 pFmD8S 45A,45B pFmD8S-M45 117G to A, 118Y to F (2) (SEQ ID NOs: 112 and 113) 2 M21pFmD8S-M45 21A, 21B pFmD8S- 117G to A, 118Y to F, 125Q to H, (SEQ IDNOs: 64 and 65) M45, 21 126M to L (4) 3 M16 pFmD8S- 16A, 16B pFmD8S-117G to A, 118Y to F, 125Q to H, M45, 21 (SEQ ID NOs: 54 and 55) M45,21, 16 126M to L, 108S to L (5) 4 M18 pFmD8S- 18A, 18B pFmD8S-2A 117G toA, 118Y to F, 125Q to H, M45, 21, 16 (SEQ ID NOs: 58 and 59) 126M to L,108S to L, 120L to M, 121M to L (7) 5 M68, M69 pFmD8S-2A 68A, 68BpFmD8S-003 117G to A, 118Y to F, 125Q to H, (SEQ ID NOs: 159 and 160)126M to L, 108S to L, 120L to M, 69A, 69B 121M to L, 162L to V, 163V toL, (SEQ ID NOs: 161 and 162) 170G to A, 171L to V (11)

TABLE 15 Generation Of pFmD8S-2B And pFmD8S-004 Constructs Mutation SiteResultant Total Mutations In Resultant Plasmid Round Introduced TemplatePrimers Plasmid With Respect to EgD8S (SEQ ID NO: 10) 1 M46 pFmD8S-M4546A, 46B pFmD8S- 117G to A, 118Y to F, 132V to L, (SEQ ID NOs: 114 and115) M45, 46 133 L to V (4) 2 M16, M21 pFmD8S- 16A, 16B pFmD8S- 117G toA, 118Y to F, 132V to L, M45, 46 (SEQ ID NOs: 54 and 55) M45, 46, 16, 21133 L to V, 108S to L, 125Q to H, 21A, 21B 126M to L (7) (SEQ ID NOs: 64and 65) 3 M18 pFmD8S- 18A, 18B pFmD8S-2B 117G to A, 118Y to F, 132V toL, M45, 46, 16, 21 (SEQ ID NOs: 58 and 59) 133 L to V, 108S to L, 125Qto H, 126M to L, 120L to M, 121M to L (9) 4 M68, M69 pFmD8S-2B 68A, 68BpFmD8S-004 117G to A, 118Y to F, 132V to L, (SEQ ID NOs: 159 and 160)133 L to V, 108S to L, 125Q to H, 69A, 69B 126M to L, 120L to M, 121M toL, (SEQ ID NOs: 161 and 162) 162L to V, 163V to L, 170G to A, 171L to V(13)

TABLE 16 Generation Of pFmD8S-3A, pFmD8S-3B And pFmD8S-005 ConstructsMutation Site Resultant Total Mutations In Resultant Plasmid RoundIntroduced Template Primers Plasmid With Respect to EgD8S (SEQ ID NO:10) 1 M49 pFmD8S 49A, 49B pFmD8S-M49 297F to V, 298V to L (2) (SEQ IDNOs: 120 and 121) 2 M26 pFmD8S-M49 26A, 26B pFmD8S- 297F to V, 298V toL, 293L to M (3) (SEQ ID NOs: 74 and 75) M49, 26 3A M61 pFmD8S- 61A, 61BpFmD8S-3A 297F to V, 298V to L, 293L to M, M49, 26 (SEQ ID NOs: 145 and146) 239F to I, 240I to F (5) 3B M62, M63 pFmD8S- 62A, 62B pFmD8S-3B297F to V, 298V to L, 293L to M, M49, 26 (SEQ ID NOs: 147 and 148) 271Lto M, 272A to S, 279T to L, 63A, 63B 280L to T (7) (SEQ ID NOs: 149 and150) 4 M63 pFmD8S-3A 63A, 63B pFmD8S-005 297F to V, 298V to L, 293L toM, (SEQ ID NOs: 149 and 150) 239F to I, 240I to F, 279T to L, 280L to T(7)

TABLE 17 Generation Of pFmD8S-4 And pFmD8S-006 Constructs Mutation SiteResultant Total Mutations In Resultant Plasmid Round Introduced TemplatePrimers Plasmid With Respect to EgD8S (SEQ ID NO: 10) 1 M51B pFmD8S 51A,51B pFmD8S-M51 346I to V, 347I to L, 348T to S (3) (SEQ ID NOs: 126 and125) 2 M15 pFmD8S-M51 15A, 15B pFmD8S- 346I to V, 347I to L, 348T to S,(SEQ ID NOs: 52 and 53) M51, 15 422L to Q (4) 3 M14 pFmD8S- 14A, 14BpFmD8S- 346I to V, 347I to L, 348T to S, M51, 15 (SEQ ID NOs: 50 and 51)M51, 15, 14 422L to Q, 416Q to V, 417P to Y (6) 4 M12 pFmD8S- 12A, 12BpFmD8S-4 346I to V, 347I to L, 348T to S, M51, 15, 14 (SEQ ID NOs: 46and 47) 422L to Q, 416Q to V, 417P to Y, 407A to S, 408V to Q (8) 5 M70pFmDBS-4 70, 70B pFmD8S-006 346I to V, 347I to L, 348T to S, (SEQ IDNOs: 163 and 164) 422L to Q, 416Q to V, 417P to Y, 407A to S, 408V to Q,418A to G, 419G to A (10)

After each round of mutagenesis, the mutations in each resulting plasmidwas confirmed by DNA sequencing. Additionally, the Δ8 desaturaseactivity of each mutant EgD8S within each mutant plasmid was comparedwith the Δ8 desaturase activity of the synthetic codon-optimized EgD8Swithin pFmD8S by transforming the plasmids into strain Y4001 (Example 3)and assaying activity based on the methodology described in Example 5.Based on these functional analyses, it was demonstrated that themutations in all 24 of the mutant EgD8S genes within the resultantplasmids generated in Tables 11, 12, 13, 14 and 15 did not affect Δ8desaturase activity (i.e., pFmD8S-M3; pFmD8S-M3,1; pFmD8S-M3,1,2;pFmD8S-1; pFmD8S-001; pFmD8S-M45; pFmD8S-M45,21; pFmD8S-M45,21,16;pFmD8S-2A; pFmD8S-003; pFmD8S-M45,46; pFmD8S-M45,46,16,21; pFmD8S-2B;pFmD8S-004; pFmD8S-M49; pFmD8S-M49,26; pFmD8S-3A; pFmD8S-3B; pFmD8S-005;pFmD8S-M51; pFmD8S-M51,15; pFmD8S-M51,15,14; pFmD8S-4; and pFmD8S-006).

Example 7 Generation of Complex Construct pFmD8S-5B by Digestion andLigation of Multiple Parent Plasmids

Plasmid pFmD8S-5B contained 16 mutant amino acids within the first halfof EgD8S. This plasmid was generated by 3-way ligation, wherein the 318bp Nco I/Bgl II fragment from pFmD8S-1 (containing 7 amino acidmutations, corresponding to M1, M2, M3 and M8) and the 954 bp Bgl II/NotI fragment from pFmD8S-2B (containing 9 amino acid mutations,corresponding to M16, M18, M21, M45 and M46) were used to replace theNco I/Not I fragment of pFmD8S (Example 4; FIG. 6D). DNA sequenceconfirmed that pFmD8S-5B contained the expected 16 amino acid mutationswithin EgD8S.

The synthesis of plasmid pFmD8S-5B is schematically diagrammed in FIG. 7(and a similar format is used in FIGS. 8 and 9). For clarity, the pFmD8Svector backbone in which each mutant EgD8S is contained is not includedwithin the figure; instead, only the 1272 bases corresponding to themutant EgD8S are shown (wherein the coding sequence for the Δ8desaturase corresponds to nucleotide bases 2-1270). Thus, the mutantEgD8S fragment labeled as “Mutant EgD8S-1” in FIG. 7 corresponds to themutant EgD8S found within plasmid pFmD8S-1 and the mutant EgD8S fragmentlabeled as “Mutant EgD8S-2B” in FIG. 7 corresponds to the mutant EgD8Sfound within plasmid pFmD8S-2B.

Similarly, the Nco I and Not I restriction enzyme sites that flank eachmutant EgD8S gene are not included in the figure. The Nco I nucleotiderecognition sequence (“CCATGG”) corresponds to the −2 to +4 region ofthe mutant EgD8S, wherein the ‘A’ position of the ‘ATG’ translationinitiation codon is designated as +1; the first nucleotide recognized aspart of the Not I nucleotide recognition sequence is nucleotide +1271 ofmutant EgD8S, wherein the ‘TAA’ STOP codon of mutant EgD8{dot over (S)}is located at +1269 to +1270.

Mutation sites are labeled on each mutant EgD8S. Those mutation sitesshown with an asterisk correspond to a single amino acid mutation (i.e.,M2*corresponds to a mutation of 12T to V), while those lacking anasterisk correspond to two individual amino acid mutations (i.e., M1corresponds to mutations 4S to A and 5K to S); those mutation sitesshown with 2 asterisks correspond to a triple amino acid mutation (i.e.,M51** corresponds to mutations 3461 to V, 3471 to L and 348T to S).

The Δ8 desaturase activity of mutant EgD8S-5B within pFmD8S-5B wascompared with the Δ8 desaturase activity of the syntheticcodon-optimized EgD8S within pFmD8S by transforming each plasmid intostrain Y4001 (Example 3) and assaying the activity based on themethodology described in Example 5. Based on this analysis, it wasdetermined that the 16 amino acid mutations within mutant EgD8S-5B(i.e., 4S to A, 5K to S, 12T to V, 16T to K, 17T to V, 66P to Q, 67S toA, 108S to L, 120L to M, 121M to L, 125Q to H, 126M to L, 117G to A,118Y to F, 132V to L and 133 L to V, corresponding to mutation sites M1,M2, M3, M8, M16, M18, M21, M45 and M46) in pFmD8S-5B did not affect theΔ8 desaturase activity.

Example 8 Generation of pFmD8S-12, pFmD8S-13, pFmD8S-23 and pFmD8S-28Constructs by Additional Site-Directed Mutagenesis of Mutant EgD8S-5BWithin Construct pFmD8S-5B

An additional series of plasmids were generated by consecutive rounds ofcontinued site-directed mutagenesis to introduce multiple selectmutations into mutant EgD8S-5B, using pFmD8S-5B (Example 7) as thestarting template. The resulting plasmids comprising mutant EgD8Ssequences, as well as details concerning the primers used to producethese mutations, are described below in Table 18. Format and columntitles of Table 18 are the same as defined above in Example 6.

TABLE 18 Generation Of pFmD8S-12, pFmD8S-13, pFmD8S-23 And pFmD8S-28Constructs Mutation Total Mutation In Resultant Plasmid Site ResultantWith Respect to EgD8S Round Introduced Template Primers Plasmid (SEQ IDNO: 10) 1A M12, M15, pFmD8S- 12A, 12B (SEQ ID NOs: 46 and 47) pFmD8S- 4Sto A, 5K to S, 12T to V, 16T to K, M26 5B 15A, 15B (SEQ ID NOs: 52 and53) 12 17T to V, 66P to Q, 67S to A, 26A, 26B (SEQ ID NOs: 74 and 75)108S to L, 120L to M, 121M to L, 125Q to H, 126M to L, 117G to A, 118Yto F, 132V to L, 133L to V, 407A to S, 408V to Q, 422L to Q, 293L to M(20) 1B M12, M15, pFmD8S- 12A, 12B(SEQ ID NOs: 46 and 47) pFmD8S- 4S toA, 5K to S, 12T to V, 16T to K, M26, 5B 15A, 15B (SEQ ID NOs: 52 and 53)13 17T to V, 66P to Q, 67S to A, M51B 26A, 26B (SEQ ID NOs: 74 and 75)108S to L, 120L to M, 121M to L, 51A, 51B (SEQ ID NOs: 126 and 125) 125Qto H, 126M to L, 117G to A, 118Y to F, 132V to L, 133L to V, 407A to S,408V to Q, 422L to Q, 293L to M, 346I to V, 347I to L, 348T to S (23) 2AM68, M70 pFmD8S- 68A, 68B (SEQ ID NOs: 159 and 160) pFmD8S- 4S to A, 5Kto S, 12T to V, 16T to K, 12 70A, 70B (SEQ ID NOs: 163 and 164) 23 17Tto V, 66P to Q, 67S to A, 108S to L, 120L to M, 121M to L, 125Q to H,126M to L, 117G to A, 118Y to F, 132V to L, 133L to V, 407A to S, 408Vto Q, 422L to Q 293L to M, 162L to V, 163V to L, 418A to G, 419G to A(24) 2B M38, M63, pFmD8S- 38A, 38B (SEQ ID NOs: 98 and 99) pFmD8S- 4S toA, 5K to S, 12T to V, 16T to K, M68, M69, 13 63A, 63B (SEQ ID NOs: 149and 150) 28 17T to V, 66P to Q, 67S to A, M70 68A, 68B (SEQ ID NOs: 159and 160) 108S to L, 120L to M, 121M to L, 69A, 69B (SEQ ID NOs: 161 and162) 125Q to H, 126M to L, 117G to A, 70A, 70B (SEQ ID NOs: 163 and 164)118Y to F, 132V to L, 133L to V, 407A to S, 408V to Q, 422L to Q, 293Lto M, 346I to V, 347I to L, 348T to S, 54A to G, 55F to Y, 279T to L,280L to T, 162L to V, 163V to L, 170G to A, 171L to V, 418A to G, 419Gto A (33)

After each round of mutagenesis, the mutations in the resulting plasmidwere confirmed by DNA sequencing. Additionally, the Δ8 desaturaseactivity of each mutant EgD8S within each mutated plasmid was comparedwith the Δ8 desaturase activity of the synthetic codon-optimized EgD8Swithin pFmD8S by transforming the plasmids into strain Y4001 (Example 3)and assaying activity based on the methodology described in Example 5.Based on these functional analyses, it was demonstrated that the 20mutations in mutant EgD8S-12 within pFmD8S-12, the 23 mutations inmutant EgD8S-13 within pFmD8S-13, the 24 mutations in mutant EgD8S-23within pFmD8S-23 and the 33 mutations in mutant EgD8S-28 withinpFmD8S-28 did not affect the Δ8 desaturase activity.

Example 9 Generation of Complex Constructs pFmD8S-008, pFmD8S-009,pFmD8S-013 and pFmD8S-015 by Digestion and Ligation of Multiple ParentPlasmids

Plasmids pFmD8S-008 and pFmD8S-009 contained 20 and 22 mutant aminoacids within the first half of EgD8S, respectively. These plasmids weregenerated by 3-way ligation, as shown in FIGS. 8A and 8B, respectively(Figure format is identical to that described for FIG. 7 in Example 7).Specifically, the 318 bp Nco I/Bgl II fragment from pFmD8S-001(containing 9 amino acid mutations in mutant EgD8S-001 corresponding toM1, M2, M3, M8 and M38) and the 954 bp Bgl II/Not I fragment from eitherpFmD8S-003 (containing 11 amino acid mutations in mutant EgD8S-003corresponding to M16, M18, M21, M45, M68 and M69) or pFmD8S-004(containing 13 amino acid mutations in mutant EgD8S-004, correspondingto M16, M18, M21, M45, M46, M68 and M69) were used to replace the NcoI/Not I fragment of pFmD8S (Example 4; FIG. 6D) to generate mutantEgD8S-008 within pFmD8S-008 and mutant EgD8S-009 within pFmD8S-009,respectively. DNA sequence confirmed that mutant EgD8S-008 contained 20amino acid mutations and mutant EgD8S-009 contained 22 amino acidmutations, as expected.

Plasmids pFmD8S-013 and pFmD8S-015, containing 28 and 31 amino acidmutations within mutant EgD8S-013 and mutant EgD8S-015, respectively,were created using a similar 3-way ligation strategy as shown in FIGS.9A and 9B (Figure format is identical to that described for FIG. 7 inExample 7). The 639 bp Nco I/Xho I fragment from either pFmD8S-009(containing 22 amino acid mutations within mutant EgD8S-009) orpFmD8S-008 (containing 20 amino acid mutations within mutant EgD8S-008)and the 633 bp Xho I/Not I fragment from either pFmD8S-23 (Example 8,containing 6 amino acid mutations within mutant EgD8S-23, correspondingto M12, M15, M26 and M70) or pFmD8S-28 (Example 8, containing 11 aminoacid mutations within mutant EgD8S-28, corresponding to M12, M15, M26,M51B, M63 and M70) were used to replace the Nco I/Not I fragment ofpFmD8S (Example 4; FIG. 6D) to generate pFmD8S-013 and pFmD8S-015,respectively. DNA sequence confirmed that mutant EgD8S-013 and mutantEgD8S-015 contained 28 amino acid mutations and 31 amino acid mutations,respectively.

The Δ8 desaturase activity of mutant EgD8S-008 within pFmD8S-008, mutantEgD8S-009 within pFmD8S-009, mutant EgD8S-013 within pFmD8S-013 andmutant EgD8S-015 within pFmD8S-015 were compared with the Δ8 desaturaseactivity of the synthetic codon-optimized EgD8S within pFmD8S bytransforming these plasmids into strain Y4001 (Example 3) and assayingactivity based on the methodology described in Example 5. Based on thesefunctional analyses, it was demonstrated the Δ8 desaturase activity wasnot affected by the 20 mutations in mutant EgD8S-008 within pFmD8S-008(i.e., 4S to A, 5K to S, 12T to V, 16T to K, 17T to V, 66P to Q, 67S toA, 108S to L, 120L to M, 121M to L, 125Q to H, 126M to L, 54A to G, 55Fto Y, 117G to A, 118Y to F, 162L to V, 163V to L, 170G to A and 171L toV, corresponding to mutation sites M1, M2, M3, M8, M16, M18, M21, M38,M45, M68 and M69), the 22 mutations in mutant EgD8S-009 withinpFmD8S-009 (i.e., 4S to A, 5K to S, 12T to V, 16T to K, 17T to V, 66P toQ, 67S to A, 108S to L, 120L to M, 121M to L, 125Q to H, 126M to L, 54Ato G, 55F to Y, 117G to A, 118Y to F, 132V to L, 133L to V, 162L to V,163V to L, 170G to A and 171L to V, corresponding to mutation sites M1,M2, M3, M8, M16, M18, M21, M38, M45, M46, M68 and M69), the 28 mutationsin mutant EgD8S-013 within pFmD8S-013 (i.e., 4S to A, 5K to S, 12T to V,16T to K, 17T to V, 66P to Q, 67S to A, 407A to S, 408V to Q, 422L to Q,108S to L, 120L to M, 121M to L, 125Q to H, 126M to L, 293L to M, 54A toG, 55F to Y, 117G to A, 118Y to F, 132V to L, 133L to V, 162L to V, 163Vto L, 170G to A, 171L to V, 418A to G and 419G to A, corresponding tomutation sites M1, M2, M3, M8, M12, M15, M16, M18, M21, M26, M38, M45,M46, M68, M69, M70) or the 31 mutations in mutant EgD8S-015 withinpFmD8S-015 (i.e., 4S to A, 5K to S, 12T to V, 16T to K, 17T to V, 66P toQ, 67S to A, 407A to S, 408V to Q, 422L to Q, 108S to L, 120L to M, 121Mto L, 125Q to H, 126M to L, 293L to M, 54A to G, 55F to Y, 117G to A,118Y to F, 3461 to V, 3471 to L, 348T to S, 279T to L, 280L to T, 162Lto V, 163V to L, 170G to A, 171L to V, 418A to G and 419G to A,corresponding to mutation sites M1, M2, M3, M8, M12, M15, M16, M18, M21,M26, M38, M45, M51B, M63, M68, M69, M70).

FIG. 10 shows an alignment of EgD8S (SEQ ID NO:10), Mutant EgD8S-23 (SEQID NO:4; Example 8), Mutant EgD8S-013 (SEQ ID NO:6; Example 9) andMutant EgD8S-015 (SEQ ID NO:8; Example 9). The method of alignment usedcorresponds to the “Clustal W method of alignment”.

Example 10 Comparison of Δ8 Desaturase Activities Among the SyntheticCodon-Optimized EgD8S and its Mutants Upon Integration into the Yarrowialipolytica Genome

This Example describes quantitative analyses of the Δ8 desaturaseactivities of EgD8S and mutants thereof included within pFmD8S-23,pFmD8S-013 and pFmD8S-015. This comparison required each of the chimericgenes comprising EgD8S (or a mutant thereof) to be inserted into thepKO2UFkF2 vector backbone. Specifically, pKO2UFkF2 comprised a 5′ and 3′portion of the Yarrowia lipolytica Δ12 desaturase gene that was designedto target integration to this locus (although plasmid integration couldalso occur via random integration into other sites of the genome).,Thus, the activities of the chimeric genes containing the syntheticcodon-optimized EgD8S, mutant EgD8S-023, mutant EgD8S-013 and mutantEgD8S-015 in the Yarrowia genome (i.e., 1 copy) could be more fairlycompared upon integration into the Yarrowia genome, as opposed to the Δ8desaturase activity levels that were obtained upon plasmid expression(i.e., via expression in pFmD8S as 1-3 copies) and reported in previousexamples.

The components of pKO2UFkF2 are as described in Table 19 below.

TABLE 19 Components Of Plasmid pKO2UFkF2 (SEQ ID NO: 165) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And NO: 165 ChimericGene Components SwaI/BsiWI FBAINm::F.D12::Pex20, comprising: 7638-1722FBAINm: Yarrowia lipolytica FBAINm promoter (WO 2005/049805) F.D12:Fusarium moniliforme Δ12 desaturase gene (WO 2005/047485) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613) AscI/BsiWI 5′ portion of Yarrowia lipolytica Δ12 desaturasegene 2459-1722 (WO 2004/104167) EcoRI/SphI 3′ portion of Yarrowialipolytica Δ12 desaturase gene 5723-5167 (WO 2004/104167) EcoRI/PacIYarrowia Ura3 gene (GenBank Accession 5723-7240 No. AJ306421)

First, the Swa I/Not I fragment from pFmD8S (comprising the chimericFBAINm::EgD8S gene, wherein EgD8S is identified as “D8-corrected” inFIG. 6D) was used to replace the SwaI/NotI fragment of pKO2UFkF2(comprising the chimeric FBAINm::F.D12 gene) to generate constructpKO2UFm8 (FIG. 11A). The same methodology was used to replace theSwaI/NotI fragment of pKO2UFkF2 with the Swa I/Not I fragments ofpFmD8S-23, pFmD8S-013 and pFmD8S-015, respectively, thereby creatingconstructs pKO2UFm8-23, pKO2UFm8-013 and pKO2UFm8-015, respectively. Assuch, the synthetic codon-optimized EgD8S, mutant EgD8S-023, mutantEgD8S-013 and mutant EgD8S-015 were each under the control of the FBAINmpromoter and the Pex20 terminator.

Plasmids pKO2UFm8, pKO2UFm8-23, pKO2UFm8-013 and pKO2UFm8-015 weredigested with AscI/SphI, and then used for transformation of strainY4001 individually according to the General Methods. Followingtransformation, cells were plated onto MM plates and maintained at 30°C. for 2 to 3 days.

A total of 6 transformants from each transformation were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into liquid MM at 30° C. and grown with shakingat 250 rpm/min for 1 day. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC. Delta-8 desaturase activity of each Δ8 desaturase are shownbelow in Table 20; conversion efficiency was calculated as described inExample 5.

TABLE 20 Δ8 Desaturase Activity In EgD8S And Its Mutants Mutations WithRespect To EgD8S Conversion Plasmid (SEQ ID NO: 10) Efficiency pKO2UFm8none 37.9% (comprising (average) wildtype EgD8S [SEQ ID NO: 10])pKO2UFm8S-23 4S to A, 5K to S, 12T to V, 16T to K,   35%, (comprisingmutant 17T to V, 66P to Q, 67S to A, 108S to L,   35%, EgD8S-23 [SEQ IDNO: 4]) 120L to M, 121M to L, 125Q to H, 36.1%, 126M to L, 117G to A,118Y to F, 36.2%, 132V to L, 133L to V, 407A to S, 39.8%, 408V to Q,422L to Q, 293L to M,   40% 162L to V, 163V to L, 418A to G, 419G to A(24) pKO2UFm8S-013 4S to A, 5K to S, 12T to V, 16T to K, 17.8%(comprising mutant 17T to V, 66P to Q, 67S to A, 407A to S, 18.4%,EgD8S-013 [SEQ ID NO: 6]) 408V to Q, 422L to Q, 108S to L, 18.6%, 120Lto M, 121M to L, 125Q to H, 24.4%, 126M to L, 293L to M, 54A to G, 55Fto 34.4% Y, 117G to A, 118Y to F, 132V to L, 39.1% 133L to V, 162L to V,163V to L, 70.8% 170G to A, 171L to V, 418A to G and 419G to A (28)pKO2UFm8S-015 4S to A, 5K to S, 12T to V, 16T to K, 17.3%, (comprisingmutant 17T to V, 66P to Q, 67S to A, 407A to S, 19.8%, EgD8S-015 [SEQ IDNO: 8]) 408V to Q, 422L to Q, 108S to L,   20%, 120L to M, 121M to L,125Q to H, 126M 20.1% to L, 293L to M, 54A to G, 55F to Y, 29.2%, 117Gto A, 118Y to F, 346I to V, 33.5% 347I to L, 348T to S, 279T to L, 280Lto 38.5% T, 162L to V, 163V to L, 170G to A, 171L to V, 418A to G and419G to A (31)

The different conversion efficiencies observed for each specific mutantEgD8S may be attributed to a “position effect” based on the respectivelocations of each gene's integration within the Yarrowia genome. In anycase, the results demonstrate that several of the transformantscomprising mutant EgD8S-23 (SEQ ID NO:4), mutant EgD8S-013 (SEQ ID NO:6)and mutant EgD8S-015 (SEQ ID NO:8) possessed Δ8 desaturase activity thatwas at least functionally equivalent (or increased) with respect to thatof the synthetic codon-optimized EgD8S (SEQ ID NO:10).

Example 11 Generation of Yarrowia lipolytica Strains Y4031, Y4032 andY4033 to Produce about 10-13.6% DGLA of Total Lipids

The present Example describes the construction of strains Y4031, Y4032and Y4033, derived from Yarrowia lipolytica Y4001U (Example 3), capableof producing 10-13.6% DGLA (C20:3) relative to the total lipids. Thesestrains were engineered to express the Δ9 elongase/Δ8 desaturasepathway, via expression of a mutant Δ8 desaturase of the presentinvention and a Δ9 elongase.

More specifically, construct pKO2UF8289 (FIG. 11B; SEQ ID NO:181) wascreated to integrate a cluster of four chimeric genes (comprising a Δ12desaturase, two copies of the mutant EgD8-23 and one Δ9 elongase) intothe Δ12 gene locus of Yarrowia genome in strain Y4001U. ConstructpKO2UF8289 contained the following components:

TABLE 21 Description of Plasmid pKO2UF8289 (SEQ ID NO: 181) RE Sites AndNucleotides Within SEQ ID Description Of Fragment NO: 181 And ChimericGene Components AscI/BsiW I 5′ portion of Yarrowia lipolytica Δ12desaturase gene (10304-9567) (WO 2004/104167) EcoRI/Sph I 3′ portion ofYarrowia lipolytica Δ12 desaturase gene (13568-13012) (WO 2004/104167)Cla I/EcoR I LoxP::Ura3::LoxP, comprising: (1-13568) LoxP sequence (SEQID NO: 18) Yarrowia Ura3 gene (GenBank Accession No. AJ306421) LoxPsequence (SEQ ID NO: 18) PmeI/ClaI GPAT::EgD9E::Lip2, comprising:(2038-1) GPAT: Yarrowia lipolytica GPAT promoter (WO 2006/031937) EgD9E:codon-optimized Δ9 elongase gene (SEQ ID NO: 177), derived from Euglenagracilis (SEQ ID NOs: 175 and 176; U.S. Pat. application Ser. No.60/739,989; see also Example 16 herein) Lip2: Lip2 terminator sequencefrom Yarrowia Lip1 gene (GenBank Accession No. AJ012632) PmeI/PacIExp::D8-23::Pex16, comprising: (4581-2124) Exp: Yarrowia lipolyticaexport protein (EXP1) promoter (WO 2006/052870 and U.S. Pat. applicationSer. No. 11/265,761) D8-23: mutant EgD8S-23 (Example 8; SEQ ID NO: 4)Pex16: Pex16 terminator sequence of Yarrowia Pex 16 gene (GenBankAccession No. U75433) Swa I/Pme I YAT::F. D12::Oct, comprising:(7055-4581) YAT: Yarrowia lipolytica YAT1 promoter (Patent PublicationU.S. 2006/0094102-A1) F.D12: Fusarium moniliforme Δ12 desaturase gene WO2005/047485 OCT: OCT terminator sequence of Yarrowia OCT gene (GenBankAccession No. X69988) Swa I/BsiW I FBAINm::D8S-23::Pex20, comprising:(7055-9567) FBAINm: Yarrowia lipolytica FBAINm promoter (WO 2005/049805)D8S-23: mutant EgD8S-23 (Example 8; SEQ ID NO: 4) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613)

Plasmid pKO2UF8289 was digested with Asc I/Sph I, and then used fortransformation of Y. lipolytica strain Y4001U (Example 3) according tothe General Methods. The transformant cells were plated onto MMLeu mediaplates and maintained at 30° C. for 2 to 3 days. The colonies werepicked and streaked onto MMLeu selection plates at 30° C. for 2 days.These cells were then inoculated into liquid MMLeu at 30° C. and shakenat 250 rpm/min for 2 days. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of DGLA in the transformants containingthe 4 chimeric genes of pKO2UF8289, but not in the Yarrowia Y4001Ucontrol strain. Most of the selected 12 strains produced about 4 to 8%DGLA of total lipids. There were 3 strains (i.e., strains #7, #8 and#12) that produced about 11.3%, 10% and 13.6% DGLA of total lipids; theywere designated as strains Y4031, Y4031 and Y4033, respectively.

Example 12 Cloning the Euglena gracilis Δ8 Desaturase Mutant (EgD8S-23)Into A Soybean Expression Vector and Co-Expression with the Isochrysisgalbana Δ9 Elongase

The present Example describes the construction of soybean expressionvector pKR1060, suitable for use in the production of DGLA (C20:3). Thisvector was engineered to enable expression of the Δ9 elongase/Δ8desaturase pathway, via expression of a mutant Δ8 desaturase of thepresent invention and a Δ9 elongase.

Through a number of cloning steps, a NotI site was added to the 5′ endof the EgD8S-23 gene from pKO2UFm8S-23 (Table 20, Example 10) to producethe sequence set forth in SEQ ID NO:182.

Vector pKR457 (SEQ ID NO:183), which was previously described in PCTPublication No. WO 2005/047479 (the contents of which are herebyincorporated by reference), contains a NotI site flanked by the Kunitzsoybean Trypsin Inhibitor (KTi) promoter (Jofuku et al., Plant Cell1:1079-1093 (1989)) and the KTi 3′ termination region, the isolation ofwhich is described in U.S. Pat. No. 6,372,965, followed by the soyalbumin transcription terminator, which was previously described in PCTPublication No. WO 2004/071467 (Kti/NotI/Kti3′Salb3′ cassette).

The NotI fragment containing EgD8S-23 described above, was cloned intothe NotI site of pKR457 to produce pKR1058 (SEQ ID NO:184).

Plasmid pKR1058 was digested with PstI and the fragment containingEgD8S-23 was cloned into the SbfI site of pKR607 (SEQ ID NO:185),previously described in PCT Publication No. WO 2006/012325 (the contentsof which are hereby incorporated by reference) to produce pKR1060 (SEQID NO:186). In this way, EgD8S-23 is co-expressed with the Isochrysisgalbana Δ9 elongase behind strong, seed-specific promoters. A schematicdepiction of pKR1060 is shown in FIG. 12.

Example 13 Cloning the Euglena gracilis Δ8 Desaturase Mutant (EgD8S-23)into a Soybean Expression Vector and Co-Expression with the Euglenagracilis Δ9 Elongase

The present Example describes the construction of soybean expressionvector pKR1059, suitable for use in the production of DGLA (C20:3). Thisvector was engineered to enable expression of the Δ9 elongase/Δ8desaturase pathway, via expression of a mutant Δ8 desaturase of thepresent invention and a Δ9 elongase.

The Euglena gracilis Δ9 elongase (SEQ ID NO:175; U.S. Patent ApplicationNo. 60/739,989; see also Example 16 herein) was amplified witholigonucleotide primers oEugEL1-1 (SEQ ID NO:187) and oEugEL1-2 (SEQ IDNO:188) using the VentR® DNA Polymerase (Cat. No. M0254S, New EnglandBiolabs Inc., Beverly, Mass.) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pKR906 (SEQ IDNO:189).

A starting plasmid pKR72 (ATCC Accession No. PTA-6019; SEQ ID NO:190,7085 bp sequence), a derivative of pKS123 which was previously describedin PCT Publication No. WO 02/008269 (the contents of which are herebyincorporated by reference), contains the hygromycin B phosphotransferasegene (HPT) (Gritz, L. and Davies, J., Gene, 25:179-188 (1983)), flankedby the T7 promoter and transcription terminator (T7prom/hpt/T7termcassette), and a bacterial origin of replication (ori) for selection andreplication in bacteria (e.g., E. coli). In addition, pKR72 alsocontains the hygromycin B phosphotransferase gene, flanked by the 35Spromoter (Odell et al., Nature, 313:810-812 (1985)) and NOS 3′transcription terminator (Depicker et al., J. Mol. Appl. Genet.,1:561-570 (1982)) (35S/hpt/NOS3′ cassette) for selection in plants suchas soybean. pKR72 also contains a NotI restriction site, flanked by thepromoter for the α′ subunit of β-conglycinin (Beachy et al., EMBO J.,4:3047-3053 (1985)) and the 3′ transcription termination region of thephaseolin gene (Doyle et al., J. Biol. Chem. 261:9228-9238 (1986)), thusallowing for strong tissue-specific expression in the seeds of soybeanof genes cloned into the NotI site.

The gene for the Euglena gracilis Δ9 elongase was released from pKR906by digestion with NotI and cloned into the NotI site of pKR72 to producepKR1010 (SEQ ID NO:191). In some instances, pKR1010 is referred to aspKR912 but the two vectors are identical.

Plasmid pKR1058 (from Example 12, SEQ ID NO:184) was digested with PstIand the fragment containing EgD8S-23 was cloned into the SbfI site ofpKR1010 (SEQ ID NO:191), to produce pKR1059 (SEQ ID NO:192). In thisway, EgD8S-23 is co-expressed with the Euglena gracilis Δ9 elongasebehind strong, seed-specific promoters. A schematic depiction of pKR1059is shown in FIG. 13.

Example 14 Co-Expressing other Promoter/Gene/Terminator CassetteCombinations

In addition to the genes, promoters, terminators and gene cassettesdescribed herein, one skilled in the art can appreciate that otherpromoter/gene/terminator cassette combinations can be synthesized in away similar to, but not limited to, that described herein for expressionin plants (e.g., soybean). For instance, PCT Publication Nos. WO2004/071467 and WO 2004/071178 describe the isolation of a number ofpromoter and transcription terminator sequences for use inembryo-specific expression in soybean. Furthermore, PCT Publication Nos.WO 2004/071467, WO 2005/047479 and WO 2006/012325 describe the synthesisof multiple promoter/gene/terminator cassette combinations by ligatingindividual promoters, genes and transcription terminators together inunique combinations. Generally, a NotI site flanked by the suitablepromoter (such as those listed in, but not limited to, Table 22) and atranscription terminator (such as those listed in, but not limited to,Table 23) is used to clone the desired gene. NotI sites can be added toa gene of interest such as those listed in, but not limited to, Table 24using PCR amplification with oligonucleotides designed to introduce NotIsites at the 5′ and 3′ ends of the gene. The resulting PCR product isthen digested with NotI and cloned into a suitablepromoter/NotI/terminator cassette.

In addition, PCT Publication Nos. WO 2004/071467, WO 2005/047479 and WO2006/012325 describe the further linking together of individual genecassettes in unique combinations, along with suitable selectable markercassettes, in order to obtain the desired phenotypic expression.Although this is done mainly using different restriction enzymes sites,one skilled in the art can appreciate that a number of techniques can beutilized to achieve the desired promoter/gene/transcription terminatorcombination. In so doing, any combination of embryo-specificpromoter/gene/transcription terminator cassettes can be achieved. Oneskilled in the art can also appreciate that these cassettes can belocated on individual DNA fragments or on multiple fragments whereco-expression of genes is the outcome of co-transformation of multipleDNA fragments.

TABLE 22 Seed-specific Promoters Promoter Organism Promoter Referenceβ-conglycinin α′-subunit soybean Beachy et al., EMBO J. 4: 3047-3053(1985) kunitz trypsin inhibitor soybean Jofuku et al., Plant Cell 1:1079-1093 (1989) Annexin soybean WO 2004/071467 glycinin Gy1 soybean WO2004/071467 albumin 2S soybean U.S. Pat. No. 6,177,613 legumin A1 peaRerie et al., Mol. Gen. Genet. 225: 148-157 (1991) β-conglycininβ-subunit soybean WO 2004/071467 BD30 (also called P34) soybean WO2004/071467 legumin A2 pea Rerie et al., Mol. Gen. Genet. 225: 148-157(1991)

TABLE 23 Transcription Terminators Transcription Terminator OrganismReference phaseolin 3′ bean WO 2004/071467 kunitz trypsin inhibitor 3′soybean WO 2004/071467 BD30 (also called P34) 3′ soybean WO 2004/071467legumin A2 3′ pea WO 2004/071467 albumin 2S 3′ soybean WO 2004/071467

TABLE 24 EPA Biosynthetic Pathway Genes Gene Organism Reference Δ6desaturase Saprolegnia diclina WO 2002/081668 Δ6 desaturase Mortierellaalpina U.S. Pat. No. 5,968,809 elongase Mortierella alpina WO 2000/12720U.S. Pat. No. 6,403,349 Δ5 desaturase Mortierella alpina U.S. Pat. No.6,075,183 Δ5 desaturase Saprolegnia diclina WO 2002/081668 Δ15desaturase Fusarium WO 2005/047479 moniliforme Δ17 desaturaseSaprolegnia diclina WO 2002/081668 elongase Thraustochytrium WO2002/08401 aureum U.S. Pat. No. 6,677,145 elongase Pavlova sp. Pereiraet al., Biochem. J. 384: 357-366 (2004) Δ4 desaturase Schizochytrium WO2002/090493 aggregatum Δ9 elongase Isochrysis galbana WO 2002/077213 Δ9elongase Euglena gracilis U.S. Provisional Application No. 60/739,989 Δ8desaturase Euglena gracilis WO 2000/34439 U.S. Pat. No. 6,825,017 WO2004/057001 WO 2006/012325 Δ8 desaturase Acanthamoeba Sayanova et al.,FEBS castellanii Lett. 580: 1946-1952 (2006) Δ8 desaturase Pavlolvasalina WO 2005/103253 Δ8 desaturase Pavlova lutheri U.S. ProvisionalApplication No. 60/795,810

Example 15 Transformation of Somatic Soybean Embryo Cultures withSoybean Expression Vectors

Culture Conditions:

Soybean embryogenic suspension cultures (cv. Jack) were maintained in 35mL liquid medium SB196 (infra) on a rotary shaker, 150 rpm, 26° C. withcool white fluorescent lights on 16:8 hr day/night photoperiod at lightintensity of 60-85 μE/m2/s. Cultures were subcultured every 7 days totwo weeks by inoculating approximately 35 mg of tissue into 35 mL offresh liquid SB196 (the preferred subculture interval is every 7 days).

Soybean embryogenic suspension cultures were transformed with thesoybean expression plasmids by the method of particle gun bombardment(Klein et al., Nature, 327:70 (1987)) using a DuPont BiolisticPDS1000/HE instrument (helium retrofit) for all transformations.

Soybean Embryogenic Suspension Culture Initiation:

Soybean cultures were initiated twice each month with 5-7 days betweeneach initiation. Pods with immature seeds from available soybean plantswere picked 45-55 days after planting. Seeds were removed from the podsand placed into a sterilized magenta box. The soybean seeds weresterilized by shaking them for 15 min in a 5% Clorox solution with 1drop of ivory soap (i.e., 95 mL of autoclaved distilled water plus 5 mLClorox and 1 drop of soap, mixed well). Seeds were rinsed using 21-literbottles of sterile distilled water and those less than 4 mm were placedon individual microscope slides. The small end of the seed was cut andthe cotyledons pressed out of the seed coat. Cotyledons were transferredto plates containing SB199 medium (25-30 cotyledons per plate) for 2weeks, and then transferred to SB1 for 2-4 weeks. Plates were wrappedwith fiber tape and were maintained at 26° C. with cool whitefluorescent lights on 16:8 h day/night photoperiod at light intensity of60-80 μE/m2/s. After incubation on SB1 medium, secondary embryos werecut and placed into SB196 liquid media for 7 days.

Preparation of DNA for Bombardment:

A DNA fragment from soybean expression plasmid pKR1059 containing theEuglena gracilis delta-9 elongase and EgD8S-23, the construction ofwhich is described herein, was obtained by gel isolation of digestedplasmids. For this, 100 μg of plasmid DNA was used in 0.5 mL of thespecific enzyme mix described below. Plasmid was digested with AscI (100units) in NEBuffer 4 (20 mM Tris-acetate, 10 mM magnesium acetate, 50 mMpotassium acetate, 1 mM dithiothreitol, pH 7.9), 100 μg/mL BSA, and 5 mMbeta-mercaptoethanol at 37° C. for 1.5 hr. The resulting DNA fragmentswere separated by gel electrophoresis on 1% SeaPlaque GTG agarose(BioWhitaker Molecular Applications) and the DNA fragments containinggene cassettes were cut from the agarose gel. DNA was purified from theagarose using the GELase digesting enzyme following the manufacturer'sprotocol.

A 50 μL aliquot of sterile distilled water containing 1 mg of goldparticles was added to 5 μL of a 1 ug/μL DNA solution (DNA fragmentprepared as described herein), 50 μL 2.5M CaCl₂ and 20 μL of 0.1 Mspermidine. The mixture was shaken 3 min on level 3 of a vortex shakerand spun for 10 sec in a bench microfuge. The supernatant was removed,followed by a wash with 400 μL 100% ethanol and another briefcentrifugation. The 400 ul ethanol was removed and the pellet wasresuspended in 85 μL of 100% ethanol. Five μL of DNA suspension wasdispensed to each flying disk of the Biolistic PDS1000/HE instrumentdisk. Each 5 μL aliquot contained approximately 0.058 mg gold perbombardment (e.g., per disk).

Tissue Preparation and Bombardment with DNA:

Approximately 100-150 mg of seven day old embryogenic suspensioncultures was placed in an empty, sterile 60×15 mm petri dish and thedish was covered with plastic mesh. The chamber was evacuated to avacuum of 27-28 inches of mercury, and tissue was bombarded one or twoshots per plate with membrane rupture pressure set at 650 PSI. Tissuewas placed approximately 2.5 inches from the retaining/stopping screen.

Selection of Transformed Embryos and Embryo Maturation:

Transformed embryos were selected using hygromycin (hygromycin Bphosphotransferase (HPT) gene was used as the selectable marker).

Following bombardment, the tissue was placed into fresh SB196 media andcultured as described above. Six to eight days post-bombardment, theSB196 was exchanged with fresh SB196 containing either 30 mg/Lhygromycin. The selection media was refreshed weekly. Four to six weekspost-selection, green, transformed tissue was observed growing fromuntransformed, necrotic embryogenic clusters.

Transformed embryogenic clusters were removed from SB196 media to 35 mLof SB228 (SHaM liquid media; Schmidt et al., Cell Biology andMorphogenesis 24:393 (2005)) in a 250 mL Erlenmeyer flask for 2-3 weeks.Tissue cultured in SB228 was maintained on a rotary shaker at 130 rpmand 26° C. with cool white fluorescent lights on a 16:8 hr day/nightphotoperiod at a light intensity of 60-85/E/m2/s.

After maturation in flasks on SB228 media, individual embryos wereremoved from the clusters, dried and screened for alterations in theirfatty acid compositions as described supra.

Media Recipes:

SB 196 - FN Lite Liquid Proliferation Medium (per liter) MS FeEDTA -100× Stock 1 10 mL MS Sulfate - 100× Stock 2 10 mL FN Lite Halides -100× Stock 3 10 mL FN Lite P, B, Mo - 100× Stock 4 10 mL B5 vitamins (1mL/L) 1.0 mL 2,4-D (10 mg/L final concentration) 1.0 mL KNO₃ 2.83 gm(NH₄)₂SO₄ 0.463 gm asparagine 1.0 gm sucrose (1%) 10 gm pH 5.8 FN LiteStock Solutions Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100× Stock Na₂EDTA* 3.724 g 1.862 g FeSO₄—7H₂O 2.784 g 1.392 g *Add first, dissolve indark bottle while stirring 2 MS Sulfate 100× stock MgSO₄—7H₂O 37.0 g18.5 g MnSO₄—H₂O 1.69 g 0.845 g ZnSO₄—7H₂O 0.86 g 0.43 g CuSO₄—5H₂O0.0025 g 0.00125 g 3 EN Lite Halides 100× Stock CaCl₂—2H₂O 30.0 g 15.0 gKl 0.083 g 0.0715 g CoCl₂—6H₂O 0.0025 g 0.00125 g 4 FN Lite P, B, Mo100× Stock KH₂PO₄ 18.5 g 9.25 g H₃BO₃ 0.62 g 0.31 g Na₂MoO₄—2H₂O 0.025 g0.0125 g

SB1 Solid Medium (Per Liter)

1 package MS salts (Gibco/BRL-Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

31.5 g glucose

2 mL 2,4-D (20 mg/L final concentration)

pH 5.7

8 g TC agar

SB199 Solid Medium (Per Liter)

1 package MS salts (Gibco/BRL-Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

30 g Sucrose

4 ml 2,4-D (40 mg/L final concentration)

pH 7.0

2 gm Geirite

SB 166 Solid Medium (Per Liter)

1 package MS salts (Gibco/BRL-Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

60 g maltose

750 mg MgCl₂ hexahydrate

5 g activated charcoal

pH 5.7

2 g gelrite

SB 103 Solid Medium (Per Liter)

1 package MS salts (Gibco/BRL-Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

60 g maltose

750 mg MgCl2 hexahydrate

pH 5.7

2 g gelrite

SB 71-4 Solid Medium (Per Liter)

1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL-Cat. No. 21153-036)

pH 5.7

5 g TC agar

2,4-D Stock

Obtain premade from Phytotech Cat. No. D 295-concentration 1 mg/mL

B5 Vitamins Stock (Per 100 mL)

Store aliquots at −20° C.

10 g myo-inositol

100 mg nicotinic acid

100 mg pyridoxine HCl

1 g thiamine

If the solution does not dissolve quickly enough, apply a low level ofheat via the hot stir plate.

SB 228- Soybean Histodifferentiation & Maturation (SHaM) (per liter) DDIH₂O 600 mL FN-Lite Macro Salts for SHaM 10× 100 mL MS Micro Salts 1000×1 mL MS FeEDTA 100× 10 mL CaCl 100× 6.82 mL B5 Vitamins 1000× 1 mLL-Methionine 0.149 g Sucrose 30 g Sorbitol 30 g Adjust volume to 900 mLpH 5.8 Autoclave Add to cooled media (≦30° C.): *Glutamine (finalconcentration 30 mM) 4% 110 mL *Note: Final volume will be 1010 mL afterglutamine addition. Since glutamine degrades relatively rapidly, it maybe preferable to add immediately prior to using media. Expiration 2weeks after glutamine is added; base media can be kept longer w/oglutamine. FN-lite Macro for SHAM 10×- Stock #1 (per liter) (NH₄)2SO₄(ammonium sulfate) 4.63 g KNO₃ (potassium nitrate) 28.3 g MgSO₄*7H₂0(magnesium sulfate heptahydrate) 3.7 g KH₂PO₄ (potassium phosphate,monobasic) 1.85 g Bring to volume Autoclave MS Micro 1000×- Stock #2(per 1 liter) H₃BO₃ (boric acid) 6.2 g MnSO₄*H₂O (manganese sulfatemonohydrate) 16.9 g ZnSO₄*7H20 (zinc sulfate heptahydrate) 8.6 gNa₂MoO₄*2H20 (sodium molybdate dihydrate) 0.25 g CuSO₄*5H₂0 (coppersulfate pentahydrate) 0.025 g CoCl₂*6H₂0 (cobalt chloride hexahydrate)0.025 g KI (potassium iodide) 0.8300 g Bring to volume Autoclave FeEDTA100×- Stock #3 (per liter) Na₂EDTA* (sodium EDTA) 3.73 g FeSO₄*7H₂0(iron sulfate heptahydrate) 2.78 g *EDTA must be completely dissolvedbefore adding iron. Bring to Volume Solution is photosensitive.Bottle(s) should be wrapped in foil to omit light. Autoclave Ca 100×-Stock #4 (per liter) CaCl₂*2H₂0 (calcium chloride dihydrate) 44 g Bringto Volume Autoclave B5 Vitamin 1000×- Stock #5 (per liter) Thiamine*HCl10 g Nicotinic Acid 1 g Pyridoxine*HCl 1 g Myo-Inositol 100 g Bring toVolume Store frozen 4% Glutamine- Stock #6 (per liter) DDI water heatedto 30° C. 900 mL L-Glutamine 40 g Gradually add while stirring andapplying low heat. Do not exceed 35° C. Bring to Volume Filter SterilizeStore frozen* *Note: Warm thawed stock in 31° C. bath to fully dissolvecrystals.

Example 16 Identification of a Δ9 Elongase from Euglena gracilis

The present Example, disclosed in U.S. Patent Application No.60/739,989, describes the isolation of a Δ9 elongase from Euglenagracilis (SEQ ID NOs:175 and 176).

Euglena gracilis Growth Conditions, Lipid Profile and mRNA Isolation

Euglena gracilis was obtained from Dr. Richard Triemer's lab at MichiganState University (East Lansing, Mich.). From 10 mL of actively growingculture, a 1 mL aliquot was transferred into 250 mL of Euglena gracilis(Eg) Medium in a 500 mL glass bottle. Eg medium was made by combining 1g of sodium acetate, 1 g of beef extract (U126-01, Difco Laboratories,Detroit, Mich.), 2 g of Bacto® tryptone (0123-17-3, Difco Laboratories),2 g of Bacto® yeast extract (0127-17-9, Difco Laboratories) in 970 mL ofwater. After filter sterilizing, 30 mL of soil-water supernatant(15-3790, Carolina Biological Supply Company, Burlington, N.C.) wasaseptically added to give the final Eg medium. Euglena gracilis cultureswere grown at 23° C. with a 16 h light, 8 h dark cycle for 2 weeks withno agitation.

After 2 weeks, 10 mL of culture was removed for lipid analysis andcentrifuged at 1,800×g for 5 min. The pellet was washed once with waterand re-centrifuged. The resulting pellet was dried for 5 min undervacuum, resuspended in 100 μL of trimethylsulfonium hydroxide (TMSH) andincubated at room temperature for 15 min with shaking. After this, 0.5mL of hexane was added and the vials were incubated for 15 min at roomtemperature with shaking. Fatty acid methyl esters (5 μL injected fromhexane layer) were separated and quantified using a Hewlett-Packard 6890Gas Chromatograph fitted with an Omegawax 320 fused silica capillarycolumn (Supelco Inc., Cat. No. 24152). The oven temperature wasprogrammed to hold at 220° C. for 2.7 min, increase to 240° C. at 20°C./min and then hold for an additional 2.3 min. Carrier gas was suppliedby a Whatman hydrogen generator. Retention times were compared to thosefor methyl esters of standards commercially available (Nu-Chek Prep,Inc. Cat. No. U-99-A).

The remaining 2 week culture (240 mL) was pelleted by centrifugation at1,800×g for 10 min, washed once with water and re-centrifuged. Total RNAwas extracted from the resulting pellet using the RNA STAT-60™ reagent(TEL-TEST, Inc., Friendswood, Tex.) and following the manufacturer'sprotocol provided (use 5 mL of reagent, dissolved RNA in 0.5 mL ofwater). In this way, 1 mg of total RNA (2 mg/mL) was obtained from thepellet. The mRNA was isolated from 1 mg of total RNA using the mRNAPurification Kit (Amersham Biosciences, Piscataway, N.J.) following themanufacturer's protocol provided. In this way, 85 μg of mRNA wasobtained.

Euglena gracilis cDNA Synthesis, Library Construction and Sequencing

A cDNA library was generated using the Cloneminer™ cDNA LibraryConstruction Kit (Cat. No. 18249-029, Invitrogen Corporation, Carlsbad,Calif.) and following the manufacturer's protocol provided (Version B,25-0608). Using the non-radiolabeling method, cDNA was synthesized from3.2 μg of mRNA (described above) using the Biotin-attB2-Oligo(dT)primer. After synthesis of the first and second strand, the attB1adapter was added, ligated and the cDNA was size fractionated usingcolumn chromatography. DNA from fractions 7 and 8 (size ranging from˜800-1500 bp) were concentrated, recombined into pDONR™ 222 andtransformed into E. coli ElectroMAX™ DH10B™ T1 Phage-Resistant cells(Invitrogen Corporation). The Euglena gracilis library was named eeg1c.

For sequencing, clones first were recovered from archived glycerolcultures grown/frozen in 384-well freezing media plates, and replicatedwith a sterile 384 pin replicator (Genetix, Boston, Mass.) in 384-wellmicrotiter plates containing LB+75 μg/mL Kanamycin (replicated plates).Plasmids then were isolated, using the Templiphi DNA sequencing templateamplification kit method (Amersham Biosciences) following themanufacturer's protocol. Briefly, the Templiphi method usesbacteriophage φ29 DNA polymerase to amplify circular single-stranded ordouble-stranded DNA by isothermal rolling circle amplification (Dean etal., Genome Res., 11:1095-1099 (2001); Nelson et al., Biotechniques,32:S44-S47 (2002)). After growing 20 h at 37° C., cells from thereplicated plate were added to 5 μL of dilution buffer and denatured at95° C. for 3 min to partially lyse cells and release the denaturedtemplate. Templiphi premix (5 μL) was then added to each sample and theresulting reaction mixture was incubated at 30° C. for 16 h, then at 65°C. for 10 min to inactivate the φ29 DNA polymerase activity. DNAquantification with the PicoGreen® dsDNA Quantitation Reagent (MolecularProbes) was performed after diluting the amplified samples 1:3 indistilled water.

The amplified products then were denatured at 95° C. for 10 min andend-sequenced in 384-well plates, using the M13F universal primer (SEQID NO:193), and the ABI BigDye version 3.1 Prism Sequencing Kit. For thesequencing reaction, 100-200 ng of templates and 6.4 pmol of primerswere used, and the following reaction conditions were repeated 25 times:96° C. for 10 sec, 50° C. for 5 sec and 60° C. for 4 min. Afterethanol-based cleanup, cycle sequencing reaction products were resolvedand detected on Perkin-Elmer ABI 3730xl automated sequencers.

Identification of Long-Chain Polyunsaturated Fatty Acid ElongationEnzyme Homologs From Euglena gracilis cDNA Library eeg1c

cDNA clones encoding long-chain polyunsaturated fatty acid elongationenzyme homologs (LC-PUFA ELO homologs or Δ9 elongases) were identifiedby conducting BLAST (Basic Local Alignment Search Tool; Altschul et al.,J. Mol. Biol. 215:403-410 (1993)) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL and DDBJ databases). TheEuglena gracilis cDNA sequences obtained above were analyzed forsimilarity to all publicly available DNA sequences contained in the “nr”database using the BLASTN algorithm provided by the National Center forBiotechnology Information (NCBI). The DNA sequences were translated inall reading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXalgorithm (Gish and States, Nat. Genet., 3:266-272 (1993)) provided bythe NCBI. For convenience, the P-value (probability) of observing amatch of a cDNA sequence to a sequence contained in the searcheddatabases merely by chance as calculated by BLAST are reported herein as“pLog” values, which represent the negative of the logarithm of thereported P-value. Accordingly, the greater the pLog value, the greaterthe likelihood that the cDNA sequence and the BLAST “hit” representhomologous proteins.

The BLASTX search using the nucleotide sequences from cloneeeg1c.pk001.n5.f revealed similarity of the protein encoded by the cDNAto the long-chain PUFA elongation enzyme from Isochrysis galbana (SEQ IDNO:173) (GenBank Accession No. AAL37626 (GI 17226123), locus AAL37626,CDS AF390174; Qi et al., FEBS Lett. 510(3):159-165 (2002)). The sequenceof a portion of the cDNA insert from clone eeg1c.pk001.n5.f is shown inSEQ ID NO:194 (5′ end of cDNA insert). Additional sequence was obtainedfrom the 3′ end of the cDNA insert of eeg1c.pk001.n5.1 as describedabove, but using the poly(A) tail-primed WobbleT oligonucleotides.Briefly, the WobbleT primer is an equimolar mix of 21 mer poly(T)A,poly(T)C, and poly(T)G, used to sequence the 3′ end of cDNA clones.

The 3′ end sequence is shown in SEQ ID NO:195. Both the 5′ and 3′sequences were aligned using Sequencher™ (Version 4.2, Gene CodesCorporation, Ann Arbor, Mich.) and the resulting sequence for the cDNAis shown in SEQ ID NO:196 (1201 bp). Sequence for the coding sequencefrom the cDNA in eeg1c.pk001.n5.f and the corresponding deduced aminoacid sequence is shown in SEQ ID NO:175 (777 bp) and SEQ ID NO:176 (258amino acids), respectively.

The amino acid sequence set forth in SEQ ID NO:176 was evaluated byBLASTP, yielding a pLog value of 38.70 (E value of 2e-39) versus theIsochrysis galbana sequence (SEQ ID NO:173). The Euglena gracilis Δ9elongase is 39.4% identical to the Isochrysis galbana Δ9 elongasesequence using the Jotun Hein method. Sequence percent identitycalculations performed by the Jotun Hein method (Hein, J. J., Meth.Enz., 183:626-645 (1990)) were done using the MegAlign™ v6.1 program ofthe LASERGENE™ bioinformatics computing suite (DNASTAR™ Inc., Madison,Wis.) with the default parameters for pairwise alignment (KTUPLE=2). TheEuglena gracilis Δ9 elongase is 31.8% identical to the Isochrysisgalbana Δ9 elongase sequence using the Clustal V method. Sequencepercent identity calculations performed by the Clustal V method(Higgins, D. G. and Sharp, P. M., Comput. Appl. Biosci., 5:151-153(1989); Higgins et al., Comput. Appl. Biosci., 8:189-191 (1992)) weredone using the MegAlign™ v6.1 program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.) with the defaultparameters for pairwise alignment (KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5 and GAP LENGTH PENALTY=10). BLAST scores andprobabilities indicate that the nucleic acid fragment described hereinas SEQ ID NO:175 encodes an entire Euglena gracilis Δ9 elongase.

Example 17 Functional Analysis of EgD8S-23 and the Euglena gracilisDelta-9 Elongase in Somatic Soybean Embryos Transformed with SoybeanExpression Vector pKR1059

Mature somatic soybean embryos are a good model for zygotic embryos.While in the globular embryo state in liquid culture, somatic soybeanembryos contain very low amounts of triacylglycerol or storage proteinstypical of maturing, zygotic soybean embryos. At this developmentalstage, the ratio of total triacylglyceride to total polar lipid(phospholipids and glycolipid) is about 1:4, as is typical of zygoticsoybean embryos at the developmental stage from which the somatic embryoculture was initiated. At the globular stage as well, the mRNAs for theprominent seed proteins, α′-subunit of β-conglycinin, kunitz trypsininhibitor 3, and seed lectin are essentially absent. Upon transfer tohormone-free media to allow differentiation to the maturing somaticembryo state, triacylglycerol becomes the most abundant lipid class. Aswell, mRNAs for α′-subunit of β-conglycinin, kunitz trypsin inhibitor 3and seed lectin become very abundant messages in the total mRNApopulation. On this basis, the somatic soybean embryo system behavesvery similarly to maturing zygotic soybean embryos in vivo, and is thusa good and rapid model system for analyzing the phenotypic effects ofmodifying the expression of genes in the fatty acid biosynthesis pathway(see PCT Publication No. WO 2002/00904, Example 3). Most importantly,the model system is also predictive of the fatty acid composition ofseeds from plants derived from transgenic embryos.

Fatty Acid Analysis of Transgenic Somatic Soybean Embryos ExpressingpKR1059

Individual single, matured, somatic soybean embryos that had beentransformed with pKR1059 (as described in Example 15 transformants werematured on SHaM liquid media) were picked into glass GC vials and fattyacid methyl esters were prepared by transesterification. Fortransesterification, 50 μL of trimethylsulfonium hydroxide (TMSH) and0.5 mL of hexane were added to the embryos in glass vials and incubatedfor 30 min at room temperature while shaking. Fatty acid methyl esters(5 μL injected from hexane layer) were separated and quantified using aHewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fusedsilica capillary column (Catalog #24152, Supelco Inc.). The oventemperature was programmed to hold at 220° C. for 2.6 min, increase to240° C. at 20° C./min and then hold for an additional 2.4 min. Carriergas was supplied by a Whatman hydrogen generator. Retention times werecompared to those for methyl esters of standards commercially available(Nu-Chek Prep, Inc.). Routinely, 6 embryos per event were analyzed byGC, using the methodology described above.

Embryo fatty acid profiles for 31 individual events (6 embryos each)containing pKR1059 were obtained and of these, 23 events contained atleast 1 embryo with greater than 1% EDA. The lipid profiles of somaticsoybean embryos expressing EgD8S-23 and the Euglena gracilis delta-9elongase for the top 5 events are shown in FIG. 14. Fatty acids areidentified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid),LA, GLA, ALA, EDA, DGLA, ERA and ETA; and, fatty acid compositionslisted in FIG. 4 are expressed as a weight percent (wt. %) of totalfatty acids. The activity of EgD8S-23 is expressed as percentdesaturation (% desat), calculated according to the following formula:([product]/[substrate+product])*100.

More specifically, the combined percent desaturation for EDA and ERA isshown as “C20% delta-8 desat”, determined as:([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100. This is also referred to as theoverall % desaturation. The individual omega-6 delta-8 desaturation(“EDA % delta-8 desat.”) was calculated as: ([DGLA]/[DGLA+EDA])*100.Similarly, the individual omega-3 delta-8 desaturation (“ERA % delta-8desat.”) was calculated as: ([ETA]/[ETA+ERA])*100. The ratio of delta-8desaturation for omega-6 versus omega-3 substrates (“ratio [EDA/ERA]%desat.”) was obtained by dividing the EDA % delta-8 desaturation by theERA % delta-8 desaturation.

In summary of FIG. 14, EgD8S-23 worked in soybean to convert both EDAand ERA to DGLA and ETA, respectively. The line with the highest averageDGLA content (i.e., 2063-1-4) had embryos with an average DGLA contentof 13.2% and an average ETA content of 4.2%. The highest DGLA and ETAcontent for an individual embryo from this line was 13.5% and 4.3%,respectively. The highest average overall % desaturation was 68.6% withthe highest overall % desaturation for an individual embryo being 69.7%.When broken down into % desaturation for the omega-6 and omega-3substrates, the highest average % desaturation was 65.4% and 81.3% forEDA and ERA, respectively. The highest % desaturation for an individualembryo was 66.6% and 84.1% for EDA and ERA, respectively. In thisexample, TegD8S-23 had a preference for ERA over EDA, with the averagedesaturation ratio ranging from 0.7 to 0.8. No significant levels of GLAwas found to accumulate in the embryos.

1. An isolated polynucleotide comprising: (a) a nucleotide sequenceencoding a mutant polypeptide having Δ8 desaturase activity, having theamino acid sequence as set forth in SEQ ID NO:198 and wherein SEQ IDNO:198 is not identical to SEQ ID NO:10; or, (b) a complement of thenucleotide sequence of part (a), wherein the complement and thenucleotide sequence of part (a) consist of the same number ofnucleotides and are 100% complementary.
 2. The isolated polynucleotideof claim 1 wherein the nucleotide sequence comprises nucleotide bases2-1270 of SEQ ID NO:197 and wherein SEQ ID NO:197 is not identical toSEQ ID NO:9.
 3. A recombinant construct comprising the isolatedpolynucleotide of claim 1 operably linked to at least one regulatorysequence.
 4. A transformed cell comprising the isolated polynucleotideof claim 1, wherein said transformed cell is selected from the groupconsisting of a plant cell and a microbial cell.
 5. The cell of claim 4wherein said cell is a yeast.
 6. The cell of claim 5 wherein the yeastis an oleaginous yeast producing at least 25% of its dry cell weight asoil.
 7. The cell of claim 6 wherein the oleaginous yeast is selectedfrom the group consisting of: Yarrowia, Candida, Rhodotorula,Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
 8. The cell ofclaim 7 wherein the cell is a Yarrowia lipolytica.
 9. A method formaking long-chain polyunsaturated fatty acids in a yeast cellcomprising: (a) providing the yeast cell according to claim 5; and (b)growing the yeast cell of (a) under conditions wherein long-chainpolyunsaturated fatty acids are produced.
 10. The method according toclaim 9 wherein the yeast is oleaginous yeast producing at least 25% ofits dry cell weight as oil.
 11. A method according to claim 10 whereinthe yeast is a Yarrowia sp.
 12. An oleaginous yeast producing at least25% of its dry cell weight as oil comprising: (a) a recombinant DNAconstruct comprising an isolated polynucleotide encoding the Δ8desaturase polypeptide according to claim 1, operably linked to at leastone regulatory sequence; and, (b) at least one additional recombinantDNA construct comprising an isolated polynucleotide operably linked toat least one regulatory sequence, the construct encoding a polypeptideselected from the group consisting of: Δ4 desaturase, Δ5 desaturase, Δ6desaturase, Δ9 desaturase, Δ12 desaturase, Δ15 desaturase, Δ17desaturase, Δ9 elongase, C_(14/16) elongase, C_(16/18) elongase,C_(18/20) elongase and C_(20,22) elongase.
 13. The yeast of claim 12selected from the group consisting of: Yarrowia, Candida, Rhodotorula,Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
 14. The yeastof claim 13 wherein the yeast cell is a Yarrowia sp. and the oilcomprises a long-chain polyunsaturated fatty acid selected from thegroup consisting of: arachidonic acid, eicosadienoic acid,eicosapentaenoic acid, eicosatetraenoic acid, eicosatrienoic acid,dihomo-gamma-linolenic acid, docosapentaenoic acid and docosahexaenoicacid.
 15. The oleaginous yeast of claim 12 wherein the Δ8 desaturasepolypeptide has the amino acid sequence as set forth in SEQ ID NO:2 andwherein SEQ ID NO:2 is not identical to SEQ ID NO:10.
 16. A method forproducing a polyunsaturated fatty acid comprising: (a) providing anoleaginous yeast comprising: (i) a recombinant construct encoding a Δ8desaturase polypeptide having the amino acid sequence as set forth inSEQ ID NO:198, wherein SEQ ID NO:198 is not identical to SEQ ID NO:10;and, (ii) a source of substrate fatty acid selected from the groupconsisting of eicosadienoic acid and eicosatrienoic acid; (b) growingthe yeast of step (a) under conditions wherein the recombinant constructencoding the Δ8 desaturase polypeptide is expressed and the substratefatty acid is converted to product fatty acid, wherein eicosadienoicacid is converted to dihomo-gamma-linolenic acid and eicosatrienoic acidis converted to eicosatetraenoic acid, and; (c) optionally recoveringthe product polyunsaturated fatty acid of step (b).
 17. A method for theproduction of dihomo-gamma-linolenic acid comprising: (a) providing ayeast cell comprising: (i) a recombinant DNA construct comprising theisolated polynucleotide of claim 1 operably linked to at least oneregulatory sequence, and; (ii) at least one additional recombinant DNAconstruct comprising an isolated polynucleotide encoding a Δ9 elongasepolypeptide operably linked to at least one regulatory sequence; (b)providing the yeast cell of (a) with a source of linolenic acid, and;(c) growing the yeast cell of (b) under conditions whereindihomo-gamma-linolenic acid is formed.
 18. The isolated polynucleotideof claim 1 comprising: (a) a nucleotide sequence encoding a mutantpolypeptide having Δ8 desaturase activity, having the amino acidsequence as set forth in SEQ ID NO:2 and wherein SEQ ID NO:2 is notidentical to SEQ ID NO:10; or, (b) a complement of the nucleotidesequence of part (a), wherein the complement and the nucleotide sequenceof part (a) consist of the same number of nucleotides and are 100%complementary.
 19. The isolated polynucleotide of claim 18 wherein thenucleotide sequence comprises nucleotide bases 2-1270 of SEQ ID NO:1 andwherein SEQ ID NO:1 is not identical to SEQ ID NO:9.